FR2776294A1 - New type II porcine circovirus - Google Patents

Abstract

The invention relates to new strains of porcine circovirus isolated from pulmonary or ganglionic samples from farms affected by the generalized wasting syndrome of post-weaning (in English PMWS). It relates to purified preparations of these strains, conventional attenuated or inativated vaccines, recombinant live vaccines, plasmid vaccines and subunit vaccines, as well as reagents and diagnostic methods. It also relates to DNA fragments which can be used for the production of subunits in an expression vector in vitro or as sequences to be integrated into an expression vector in vivo of virus or plasmid type.

Description

The present invention relates to novel strains of porcine circovirus (PCV for Porcine CircoVirus) responsible for PMWS syndrome (Porcine Multis ys temic Lasting Syndrome or Post-Weaning Multis ys temic
Wasting Syndrome), reagents and methods for their detection, vaccination methods and vaccines, and methods for the production of these reagents and vaccines.

PCV was originally detected as a non-cytopathogenic contaminant in PK / 15 pig kidney cell lines. This virus has been classified as Circoviridae with Chicken Anemia Virus (CAV for Chicken
Anemia Virus) and PBVDV (Pscittacin Beak and Feather Disease Virus).

 These are small, non-enveloped (15 to 24 nm) viruses, the common feature of which is that they contain a genome in the form of a 1.76 to 2.31 kb circular single-stranded DNA. This genome was initially thought to encode a polypeptide of about 30 kDa (Todd et al., Arch Virol 1991, 117: 129135). Recent work, however, has shown a more complex transcription (Meehan B.M. et al., 1997, 78: 221-227). Furthermore, no significant nucleotide sequence homology or antigenic determinants common to all three types of known circovirus are known.

PCV from PK / 15 cells is considered non-pathogenic. The sequence is known from BM Meehan et al., J Gen
Virol 1997 (78) 221-227. Only very recently have authors thought that strains of PCV could be pathogenic and associated with PMWS syndrome (Gupi PS Nayar et al., Can Vet J, Vol 38, 1997: 385387 and Clark EG, Proc Am Assoc Swine Prac 1997: 499-501). Nayar et al. detected PCV DNA in pigs with PMWS syndrome by PCR techniques. However, no wild PCV strain has been isolated and purified to date.

 The PMWS syndrome detected in Canada, the United States and France is clinically characterized by progressive weight loss and manifestations such as tachypnea, dyspnea and jaundice. Pathologically, it results in lymphocytic or granulomatous infiltrations, lymphadenopathies and, more rarely, lymphocytic or granulomatous hepatitis and nephritis (Clark E. G., Proc. Am.

Assoc. Swine Prac. 1997: 499-501; Veterinary Week No. 26, supplement to Veterinary Week 1 996 (834), Veterinary Week 1997 (857): 54, Gupi P. S. Nayar et al., Can Vet J, 38, 1997: 385387).

 The applicant has succeeded in isolating five new strains under PCV from lung or ganglionic samples from farms located in Canada, the United States (California) and France (Brittany), hereinafter called circoviruses according to the invention. . These viruses have been detected in pig lesions with PMWS syndrome, but not in healthy pigs.

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

 The subject of the present invention is therefore porcine circovirus group II, as defined above, isolated or in the form of a purified preparation.

 The invention relates to any porcine circovirus capable of being isolated from a physiological sample or from a tissue sample, in particular lesions, from a sick pig presenting the PMWS syndrome, in particular by following the method described in the examples, in particular circovirus type II.

The present invention more particularly relates to purified preparations of five strains, which have been deposited with ECACC (European
Collection of Cell Cultures, Center for Applied Microbiology & Research,
Porton Down, Salisbury, Wiltshire SP4 OJG, United Kingdom) on Thursday 2 October 1997: - Provisional Access No. V97100219 (hereinafter referred to as lmp.1008PCV) - Provisional Access No. V97100218 (hereinafter referred to as lmp.1010PCV) - nO temporary access V97100217 (here called lmp.999PCV). and, on Friday, January 16, 1998: - Provisional Access No. V98 011608 (here referred to as Imp 1011-48285) - Provisional Access No. V98 011609 (here referred to as Imp 1011-48121)
The invention is intended to consider porcine circoviruses isolated from a diseased pig and / or circoviruses having a significant serological kinship with the strains of the invention and / or the circoviruses having a cross-hybridization with the strains of the invention under conditions of stringency such that there is no hybridization with the PCV PK / 15 strain.

 Viral strains isolated from a physiological sample or from a tissue sample, in particular from a lesion, of a pig presenting the PMWS syndrome can be advantageously propagated on cell lines such as, in particular, porcine kidney cell lines, in particular particularly PK / 15 free from contamination (in particular for PCV, as well as for pestiviruses, porcine adenovirus and porcine parvovirus) with a view to their multiplication or specifically for the production of whole antigen (eg viruses) and / or subunits (eg polypeptides).

 Very remarkably and unexpectedly, these isolates have proved very productive in PK / 15 culture, which has undeniable advantages for the production of viruses or antigens, in particular for the production of inactivated vaccine.

 The subject of the present invention is also the circovirus preparations isolated after passages on cells, in particular cell lines, eg PK / 15 cells, cultured in vitro by being infected with at least one of the circoviruses according to the invention or of any porcine circovirus. likely to be isolated from a physiological sample or tissue sample, including lesions, from a pig with the PMWS syndrome. It also relates to the supernatants or culture extracts, optionally purified by standard techniques, and generally any antigenic preparation obtained from in vitro cultures.

 The subject of the invention is also the immunogenic active principles and the vaccines containing at least one antigen as defined above.

 It may be immunogenic active principles based on attenuated whole live viruses, or vaccines prepared with these active principles, the attenuation being carried out according to the usual methods, eg by passage over cells, preferably by passage over pork cells. , in particular lines, such as PK / 15 cells (for example from 50 to 150, in particular of the order of 100, passages). These vaccines generally comprise a veterinarily acceptable carrier or diluent, optionally a veterinarily acceptable adjuvant as well as optionally a lyophilization stabilizer.

 These vaccines will preferably comprise from 103 to 106 TClD5O.

 It may also be immunogenic active ingredients or vaccines based on circovirus antigen according to the invention, in the inactivated state. The vaccine further comprises a veterinarily acceptable carrier or diluent, optionally with additionally a veterinarily acceptable adjuvant.

 The circoviruses according to the invention, with the fractions that may be present, are inactivated according to the techniques known to those skilled in the art.

Inactivation will preferably be effected chemically, e.g. by exposing the antigen to a chemical agent such as formaldehyde (formaldehyde), paraformaldehyde, il-propiolactone or ethylene imine or its derivatives. The preferred inactivation method will here be exposure to a chemical agent and in particular to ethylene imine or β-propiolactone.

Preferably, the inactivated vaccines according to the invention will be added, advantageously by being presented in the form of emulsions, for example water-in-oil or oil-in-water, according to the well known techniques of
The skilled person. The adjuvant nature may also come from the incorporation into the active ingredient of a usual adjuvant compound.

Among the adjuvants that may be used include, for example, alumina hydroxide, saponins (eg 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, 210), Avridine (Vaccine Design p. 148), DDA (Dimethyldioctadecylammonium bromide, Vaccine Design p.
Polyphosphazene (Vaccine Design p 204), or oil-in-oil emulsions
Water based on mineral oil, squalane (eg SPT emulsion, Vaccine Design p 147), squalene (eg MF59, Vaccine Design p 183), or water-in-oil with metabolizable oil ( preferably according to WO-A-94 20071) as well as the emulsions described in US Pat. No. 5,422,109. It is also possible to choose combinations of adjuvants, for example Avridinee or DDA associated with an emulsion.

 These vaccines will preferably comprise from 10 6 to 10 8 TClD50.

 The adjuvants of the live vaccine may be selected from those given for the inactivated vaccine. Emulsions will be preferred. To those indicated for the inactivated vaccine, one can add those described in WO-A-9416681.

As a freeze-drying stabilizer, there may be mentioned by way of example
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 alkali metal phosphates.

 The vaccines according to the invention may comprise one or more active principles (antigens) of one or more (2 or 3) circoviruses according to the invention.

 The invention also provides for combining vaccination against porcine circovirus with vaccination against other porcine pathogens, particularly those that may be associated with PMWS syndrome. The vaccine according to the invention may therefore comprise another valence corresponding to another pathogen of the pig.

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

The subject of the present invention is therefore a DNA fragment containing
all or part of one of these sequences. It goes without saying that the invention covers
automatically the equivalent sequences, that is to say the sequences do not
not changing the functionality or strain specificity of the sequence
described nor polypeptides encoded by this sequence. Will be heard
included the sequences differing by degeneracy of the code.

 The invention also covers the equivalent sequences in that they are capable of hybridizing to the above sequence under high stringency conditions and / or have a high homology with the strains of the invention and belong to the group II defined upper.

These sequences and their fragments can advantageously be used for the expression in vitro or in vivo of polypeptides with the aid of
appropriate vectors.

 In particular, open reading frames that can be used for this purpose have been identified on the genomic sequence of type II circoviruses. The invention relates to any polypeptide containing at least one of these open reading frames. Preferably, the invention relates to a protein consisting essentially of COL4, COL7, COL10 or COL13.

For the expression of subunits in vitro, as a means of expression, preference will be given to E. coli or baculovirus (US Pat. No. 4,745,051). The coding sequence (s) or fragments thereof are integrated into the baculovirus genome (eg Baculovirus Autographa californica Nuclear Polyhedrosis Virus
AcNPV) and the latter is then propagated on insect cells, eg Spodoptera frugiperda Sf9 (ATCC deposit CRL 1711). Subunits can still be produced in eukaryotic cells such as yeasts (eg Saccharomyces cerevisiae) or mammalian cells (eg CHO, BHK).

 The subject of the invention is also the polypeptides which will be produced in vitro by these means of expression, and then optionally purified according to conventional techniques. It also relates to a subunit vaccine comprising at least one polypeptide as obtained, or fragment, in a vehicle or diluent acceptable veterinary and possibly a veterinarily acceptable adjuvant.

 For expression in vivo for the production of recombinant live vaccines, the coding sequence (s) or their fragments are inserted in an appropriate expression vector under conditions permitting the expression of the polypeptide (s). Suitable vectors may be live viruses, preferably capable of multiplication in pigs, non-pathogenic for pig (naturally non-pathogenic or rendered such), according to techniques well known to those skilled in the art. In particular, it will be possible to use porcine herpesviruses such as Aujeszky's disease virus, porcine adenovirus, poxviruses, in particular vaccinia virus, avipox, canarypox or swinepox. Plasmid DNAs can also be used as vectors (WO-A-90 11092, WO-A-93 19813, WO-A-94 21797, WO-A-95 20660).

 The subject of the invention is thus vectors and recombinant or plasmid live vaccines (polynucleotide vaccines or DNA) thus produced, the vaccines further comprising a veterinary vehicle or diluent which is acceptable on a veterinary level.

 The subject of the present invention is also a method for inducing an immune response in pigs with respect to the circoviruses according to the invention. In particular, it relates to an effective vaccination method in pigs.

 This method provides for the administration to pigs, on one or more occasions, of a vaccine above. It is also possible to combine several types of vaccines supra in the same vaccination protocol.

 This method not only provides for adult pigs, but also for young or pregnant females. Vaccination of the latter makes it possible to confer passive immunity to newborns (maternal antibodies).

 The present invention also offers the possibility of diagnosing the presence of circoviruses according to the invention in pigs. It therefore relates to diagnostic tests and methods relating thereto using the reagents that will be described below.

 Knowing the sequences of the different circoviruses makes it possible to define common sequences that make it possible to produce reagents capable of recognizing all the known porcine circoviruses.

 Those skilled in the art may also choose fragments of sequences corresponding to regions having little or no homology with the corresponding sequence of the circovirus PK / 15 in order to be able to perform a specific diagnosis.

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

 A first reagent consists in the DNA sequences disclosed herein and their fragments, which will be used in particular as probes or primers in well-known hybridization or PCR ("Polymerase Chain Reaction") PCR techniques.

 A second reagent consists of the polypeptides encoded by these sequences from the virus or expressed using a vector (see above), or synthesized by the chemical method according to conventional peptide synthesis techniques.

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

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

 The antigens and antibodies according to the invention may be used in all known laboratory diagnostic techniques.

 However, it will be preferred to use them in techniques that can be implemented directly in the field by the veterinarian, the breeder or the owner of the animal. The skilled person has all the laboratory and field techniques and is therefore perfectly able to adapt them to the use of this antigen and / or antibodies as diagnostic reagent (s).

 The diagnostic techniques that will be preferentially used in the context of the present invention are Western blotting, immunofluorescence, ELISA and immunochromatography.

 With regard to the implementation of immunochromatography methods, the specialist will be able to refer in particular to Robert F.

Zurk et al., Clin. Chem. 31/7, 1144-1150 (1985) and to patents or patent applications WO-A-88 / 08,534, WO-A-91 / 1,2528, EP-A-291,176, EP-A299,428, 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 Pat. 5,232,835 and US-A-5,238,652.

 Thus, it is preferred to detect the specific antibodies in the sample by indirect test, by competition or by displacement. To do this, the antigen itself is used as a diagnostic reagent, or a fragment of this antigen, retaining antibody recognition. The labeling may advantageously be a peroxidase labeling or a particulate marking, preferably with colloidal gold.

 One can also seek to detect the antigen itself in the sample using a labeled antibody specific for this antigen. The marking is advantageously as described above.

By antigen-specific antibody that can be used in particular in competition or displacement or for the detection of the antigen itself, it is meant that the antigen-specific monoclonal and polyclonal antibodies, fragments of these antibodies, preferably Fab or F fragments (ab ) '2
Another aspect of the invention is the production of antibodies, polyclonal or monoclonal, specific for the antigen according to the invention, these antibodies can then be used in particular as diagnostic reagents for the detection of antigen in a sample of physiological fluid or in a tissue sample, or even for the detection of antibodies present in such a sample or sample. The invention also includes immunologically functional fragments of these antibodies, particularly fragments
F (ab) and F <ab) '2.

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

 It will be possible, in particular, to proceed, as is known per se, to the fusion of spleen cells of mice immunized with the antigen or at least one of its fragments with suitable myeloma cells.

 The subject of the invention is also a preparation, preferably pure or partially purified, or even crude, of monoclonal or polyclonal antibodies specific for the antigen, in particular mouse or rabbit antibodies.

 The present invention also makes it possible to determine epitopes of interest, in particular on the basis of the DNA sequences described here, whether they are epitopes of vaccine interest or epitopes of interest in diagnosis. From the DNA sequence of the circovirus genome according to the invention, a person skilled in the art is able to determine epitopes according to known methods, for example an appropriate computer program or PEPSCAN. Epitopes are immunodominant regions of proteins and as such are 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 can be used as diagnostic reagents.

 At a minimum, an epitope is a peptide having 8 to 9 amino acids. Generally, a minimum of 13 to 25 amino acids will be preferred.

 Those skilled in the art are therefore able, by 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.

 The subject of the invention is also a diagnostic kit comprising this antigen and / or polyclonal or monoclonal antibodies specific for this antigen. These are in particular diagnostic kits corresponding to the diagnostic techniques described above.

The invention will now be described in more detail with the aid of nonlimiting exemplary embodiments, taken with reference to the drawing, in which
Figure 1: DNA sequence of the genome of strain Imp 1011-48121
Figure 2: DNA sequence of the genome of strain Imp 1011-48285
Figure 3: DNA sequence of the genome of strain Imp 999
Figure 4: DNA sequence of the genome of strain Imp 1010
FIG. 5: alignment of the 4 sequences according to FIGS. 1 to 4 with the sequence of the PCV PK / 15 strain
Figure 6: DNA sequence of the genome of strain Imp 999 as defined in the first depot in France from October 3, 1997
Figure 7: Alignments of the sequence of Figure 6 with the sequence of the PK / 15 strain
List of SEQ ID sequences
SEQ ID NO: 1 DNA sequence of the genome of strain Imp 1011-48121
SEQ ID NO: 2 DNA sequence of the genome of strain Imp 1011-48285
SEQ ID NO: 3 DNA sequence of the genome of strain Imp 999
SEQ ID NO: 4 genome DNA sequence of strain Imp 1010
SEQ ID NO: 5 DNA sequence of the genome of strain PK / 15
SEQ ID NO: 6 DNA sequence of the genome of strain Imp 999 as defined in the first depot in France of October 3, 1997.

EXAMPLES
Example 1 Culture and Isolation of Porcine Circovirus Strains
Tissue samples were collected in France, Canada and
USA from lungs and lymph nodes of piglets. These piglets showed clinical signs typical of post-weaning generalized dieback syndrome. To facilitate virus isolation, the tissue samples were frozen at -700C immediately after autopsy.

 For virus isolation, suspensions containing approximately 15% tissue sample were prepared in minimal medium containing Earl salts (EMEM, BioWhittaker UK Ltd., Wokingham, UK), penicillin (100 μl ml) and streptomycin (100 μg / ml) (MEM-SA medium), by grinding the tissues with sterile sand using a sterile mortar and pestle. This milled preparation was then taken up in MEM-SA, then centrifuged at 3000 g for 30 minutes at + 40C to harvest the supernatant.

 Prior to inoculation of the cell cultures, a volume of 100 μl of chloroform was added to 2 ml of each supernatant 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 the supernatant harvested. This supernatant was then used as an inoculum for viral isolation experiments.

 All viral isolation studies were performed on PK / 15 cell cultures, known to be uncontaminated with 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 Fetal Material. Vet. Microbiol. 1995. 44. 49-64).

The isolation of porcine circoviruses was carried out according to the following technique:
Monolayers of PK / 15 cells were dissociated by trypsination (with a trypsin-versene mixture) from confluent cultures, and taken up in MEM-SA medium containing 15% fetal calf serum uncontaminated with pestivirus (= MEM medium). -G) in a final concentration of about 400,000 cells per ml. 10 ml aliquots of this cell suspension were then mixed with 2 ml aliquots of the inocula described above, and the final mixtures were aliquoted in 6 ml volumes into two 25 cm 2 Falcon flasks. These cultures were then incubated at + 370C for 18 hours in an atmosphere containing 10% CO2.

After incubation, the culture medium of the semi-confluent monolayers was treated with 300 mM D-glucosamine (Cat., G48175, Sigma-Aldrich
Company Limited, Poole, UK) (Tischr I. et al., Virol Arch.19879639-57), and then the incubation was continued for an additional 48-72 hours at + 37 OC. Following this last incubation, one of the two Falcons of each inoculum has undergone 3 successive cycles of freezing / thawing. Cells
PK / 15 of the remaining Falcon were treated with a trypsin-versene solution, resuspended in 20 ml of MEM-G medium, and then seeded in 75 cm 2 Falcons at a concentration of 400,000 cells / ml. The freshly seeded flasks were then "super-infected" by the addition of 5 ml of the corresponding lysate obtained after the freeze / thaw cycles.

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

 A volume of 5 ml of the "superinfected" suspension was removed and inoculated in a 55 mm diameter Petri dish containing a sterile, defatted glass slide. The flask and glass coverslip cultures were incubated at + 370C and treated with glucosamine as described in Example 1. The glass slide cultures were harvested 24 to 48 hours after the glucosamine treatment and fixed. either with acetone for 10 minutes at room temperature or with 10% buffered formaldehyde for 4 hours. Following this fixation, all the glass coverslips were stored at 700C, on silica gel, prior to their use for in situ hybridization studies and immunocytochemical staining studies.

Example 3 Techniques for Detecting PCV Sequences by In Situ Hybridization
In situ hybridization was performed on tissues taken from diseased and formaldehyde-fixed pigs and also on inoculated cell culture preparations for virus isolation (see Example 2) and fixed on glass slides.

Comprehensive genomic probes corresponding to porcine circovirus PK / 1 (PCV) and to chicken anemia virus (CAV) from the purified plasmids described above (1 μg for each probe) and from Hexanucleotide primers at random using a non-radioactive commercial labeling kit ("DIG DNA labeling kit", Boehringer Mannheim, Lewes,
UK) according to the supplier's recommendations.

 The digoxigenin-labeled probes were taken up in a volume of 50-100 μl of sterile water prior to their use for in situ hybridization.

Paraffin-embedded, formaldehyde-fixed, paraffin-embedded tissue samples and formaldehyde-fixed, infected cell cultures were prepared for the detection of nucleic acids.
PCV according to the following technique
Sections 5 μm thick were cut from paraffin-embedded tissue blocks, dewaxed, and rehydrated in successive decreasing concentration alcohol solutions. Tissue sections and formaldehyde-fixed cell cultures were respectively incubated for 15 minutes and 5 minutes at + 370C in a 0.5% Proteinase K solution in 0.05M Tris-HCl buffer, 5 mM EDTA (pH 7.6). The slides were then placed in a 1% glycine solution in autoclaved distilled water for 30 seconds, washed twice with PBS buffer (phosphate buffer saline) 0.01.
M (pH 7.2), and finally washed for 5 minutes in sterile distilled water. They were finally dried in the open air and placed in contact with the probes.

 Each tissue / probe preparation was covered with a clean, defatted coverslip, then placed in an oven at + 900C for 10 minutes, then placed in contact with an ice block for 1 minute, and finally incubated for 18 hours at + 370C. The preparations were then briefly immersed in 2X sodium citrate salt (SSC) buffer (pH 7.0) to remove the protective coverslips, then washed twice for 5 minutes in 2X SSC 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, 0.15 M NaCl (pH 7.5) (maleic buffer) for 10 minutes, and then incubated in a solution of 1% of reagent blocker (Cat. No. 10961 76, Boehringer Mannheim UK, Lewis, East Sussex, UK) in maleic buffer for 20 minutes at + 370C.

 The preparations were then incubated with a 1/250 solution of an anti-digoxigenin monoclonal antibody (Boehringer Mannheim), diluted in blocking buffer, for 1 hour at + 370C, washed in PBS and finally incubated with an anti-biotin antibody. mouse immunoglobulin for 30 minutes at + 37 OC.

The preparations were washed with PBS and the endogenous peroxidase activity was blocked by treatment with 0.5% hydrogen peroxide solution.
PBS for 20 minutes at room temperature. The preparations were washed again with PBS and treated with a 3-amino-9-diethylcarbazole (AEC) substrate (Cambridge Bioscience, Cambridge, UK) prepared extemporaneously.

After a final wash with tap water, the preparations were counterstained with hematoxylin, "blued" under city water, and mounted under microscopic lamellae with mounting liquid (GVA Mount, Cambridge
Bioscience, Cambridge, UK). Experience checks included the use of an irrelevant negative probe (CAV) and a positive probe (PCV) on samples from diseased pigs and non-diseased pigs.

Example 4 Immunofluorescence PCV Detection Technique
Initial screening of all acetone-fixed cell culture preparations was performed by an indirect immunofluorescence (IFI) technique using a 1: 100 dilution of a pool of adult pig sera. This pool of sera includes sera from 25 adult sows from Northern lreland and is known to contain antibodies against a wide variety of swine viruses, including
PCV: porcine parvovirus, porcine adenovirus, and PRRS virus. The IFI technique was performed by serum contact (diluted in PBS) with cell cultures for one hour at + 370C, followed by two washes in PBS. The cell cultures are then stained with a 1/80 dilution in PBS of a rabbit anti-pork immunoglobulin antibody conjugated to fluorescein isothiocyanate for one hour, then washed in PBS and mounted in glycerol buffer prior to microscopic observation under ultraviolet light.

Example 5 Results of In Situ Hybridization on Sick Pigs Tissues
In situ hybridization, using a PCV genomic probe, performed on tissues collected from French, Canadian and Californian piglets with generalized wasting lesions and fixed with formaldehyde, revealed the presence of PCV nucleic acids associated with the lesions. many of the lesions studied.

No signal was observed when the PCV genomic probe was used on tissues taken from non-diseased pigs or when the CAV probe was used on diseased pig tissues. The presence of PCV nucleic acid has been identified in the cytoplasm and nucleus of many mononuclear cells infiltrating lesions in the lungs of California piglets. The presence of PCV nucleic acid has also been demonstrated in pneumocytes, bronchial and bronchiolar epithelial cells, and in endothelial cells of small arterioles, venules and lymphatic vessels.

 In French sick pigs, the presence of PCV nucleic acid was detected in the cytoplasm of many follicular lymphocytes and in the intrasinusoldal mononuclear cells of the lymph nodes. PCV nucleic acid has also been detected in occasional syncytia. Based on these detection results, lung samples from California pigs, mesenteric lymph nodes from French pigs, and Canadian hog organs were selected for the purpose of isolating the new porcine circovirus strains.

EXAMPLE 6 Results of the Cell Culture of the New Porcine Circovirus Strains and Immunofluorescence Detection
No cytopathic effect (CPE) was observed in the cultures of cells inoculated with the samples taken from French pigs (strain lmp.1008), Californian (strain lmp.999) and Canadian (strain Lump.1010) showing signs Clinical signs of generalized wasting syndrome.

However, immuno-labeling of preparations from inoculated cell cultures, after fixation with acetone and with a pool of polyclonal pig sera, revealed nuclear fluorescence in many cells in cultures inoculated from the lungs of rats. calf piglets (strain lmp.999), from the mediastinal lymph nodes of French piglets (Lump.1008 strain), and from organs of Canadian piglets (lump.1010 strain).

Example 7 Extraction of Genomic DNA from Porcine Circoviruses
The replicative forms of the novel porcine circovirus (PCV) strains were prepared from infected PK / 15 cell cultures (see Example 1) (10).
75 cm 2 Falcons) harvested after 72-76 hours of incubation and treated with glucosamine, as described for cloning the replicative form of CAV (Todd.

 D and ai Dot blot hybridization assay for chicken anemia agent using a cloned DNA probe. J. Clin. Microbiol. 991. 29. 933-939). The double-stranded DNA of these replicative forms was extracted according to a modification of the Hirt technique (Hirt B.

Selective extraction of polyoma virus DNA from infected cell cultures. J. Mol. Biol.

 1 967, 36, 365-369), as described by Molitor (Molitor, T.W. et al., Porcine parvovirus DNA: characterization of the genomic and replicative form DNA of two virus isolates, Virology, 1984. 137. 241-254).

Example 8: Restriction map of the replicative form of the genome of the lmp.999 porcine circovirus strain.

The DNA (1-5 -5, sg) extracted according to the Hirt technique was treated with S1 nuclease (Amersham) according to the supplier's recommendations, and then this DNA was digested with various restriction enzymes (Boehringer Mannheim, Lewis , East
Sussex, UK) and the products of the digestion were separated by 1.5% agarose gel electrophoresis 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 the cultures of the lmp.999 strain has a unique EcoRI site, 2 SacI sites and does not have a PstI site. This restriction profile is therefore different from the restriction profile exhibited by PCV strain PK / 15 (Meehan B. et al., Sequence of porcine circovirus DNA: affinities with plant circoviruses, 1997.

78. 221-227) which has instead a Pstl site and does not have a site
EcoRI.

Example 9: Cloning of the genome of porcine circovirus strain Imp.999
The restriction fragment of about 1.8 kbp generated by digestion of the double-stranded replicative form of the PCV lmp.999 strain with the restriction enzyme EcoRI was isolated after electrophoresis on a 1.5% agarose gel ( see Example 3) using a Qiagen commercial kit (QIAEXII Gel Extraction Kit, Cat., 20021, QIAGEN Ltd., Crawley, West Sussex, UK). This restriction fragment
EcoRI-EcoRI was then ligated with the vector pGEM-7 (Promega, Medical
Supply Company, Dublin, Ireland), previously digested with the same restriction and dephosphorylated enzymes, following standard cloning techniques (Sambrook, J. et al., Molecular Cloning: A Laboratory Manual, 2nd Edition, Cold).
Spring Harbor Laboratory. Cold Spring Harbor. New York. 1989). The plasmids obtained were transformed into an Escherichia coli host strain JM 109 (Stratagene, La Jolla, USA) according to standard techniques. The EcoRI-EcoRI restriction fragment of the PCV lmp.999 strain was also cloned into the EcoRI site of the pBlueScript SK + vector (Stratagene Inc. La Jolla, USA). Among the clones obtained for each host strain, at least 2 clones containing fragments of the expected size were selected. The clones obtained were then cultured and the plasmids containing the complete genome of the lmp.999 strain were purified in small volume (2 ml) or in large volume (250 ml) according to the standard plasmid preparation and purification techniques.

Example 10 Sequencing of genomic DNA (double-stranded replicative form) of the PCV lmp.999 strain.

 The nucleotide sequence of 2 EcoRI lmp.999 clones (clones pGEM-7/2 and pGEM-7/8) was determined according to the technique of Sanger dideoxynucleotides using the "AmpliTaq DNA polymerase FS" sequencing kit (Cat et num; 402079 PE Applied Biosystems, Warrington, UK) and an Applied BioSystems AB1373A automated sequencing device as recommended by the supplier. The initial sequence reactions were made with the universal primers M13 "forward" and "reverse". The following sequence reactions were generated according to the "walking on DNA" technique. The oligonucleotides necessary for these subsequent sequencing were synthesized by Life Technologies (Inchinnan Business Park, Paisley, UK).

The generated sequences were assembled and analyzed using the MacDNASIS version 3.2 software. (Cat 22020101, Appligene, Durham, UK). The different open reading frames were analyzed using the algorithm
BLAST available on the National Center for Biotechnology Information server (NCBI, Bethesda, MD, USA).

 The complete sequence (EcoRI-EcoRI fragment) initially obtained from clone pGEM-7/8 (SEQ ID NO: 6) is presented in FIG. It begins arbitrarily after the G of the EcoRI site and presents some uncertainties in terms of nucleotides.

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

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

The genome size of these four strains is Imp 1011-48121 1767 nucleotides Imp 1011-48285 1767 nucleotides Imp 999 1768 nucleotides Imp 1010 1768 nucleotides
Example 11: Analysis of the sequence of the PCV lmp.999 strain.

 When the sequence generated from the lmp.999 strain was used for a homology search with respect to the sequences contained in the GenBank database, the only significant homology that has been detected is a homology of about 76% (at the nucleic acid level) with the sequence of strain PK / 15 (accession numbers Y09921 and U49186) (see Figure N05).

 At the amino acid level, the search for homology of the translation of the sequences in the 6 phases with the databanks (BLAST X algorithm on the NCBI server) made it possible to highlight a homology of 94% with the open reading frame corresponding to the theoretical replicase of the BBTV virus similar to plant circoviruses (GenBank identification number 1841515) encoded by GenBank sequence U49186.

 No other sequence contained in the databases shows significant homology with the sequence generated from the PCV lmp.999 strain.

 Sequence analysis obtained from the lmp.999 strain cultured from lesions taken from Californian piglets showing clinical signs of generalized wasting syndrome clearly shows that this viral isolate is a new strain of porcine circovirus.

Example 12 Comparative Sequence Analysis
The alignment of the nucleotide sequences of the 4 new PCV strains was done with the sequence of the PCV PK / 15 strain (FIG. 5). A homology matrix taking into account the four new strains and the earlier strain PK / 15 was performed. The results are as follows 1: Imp 1011-48121 2: Imp 1011-48285 3: Imp 999 4: Imp 1010 5: PK / 15

<tb><SEP> 1 <SEP> 2 <SEP> 3 <SEP> 4 <SEP> 5
<tb> 1 <SEP> 1.0000 <SEQ> 0.9977 <SEP> 0.9615 <SEP> 0.9621 <SEP> 0.7600
<tb> 2 <SEP> 1.0000 <SEP> 0.9621 <SEP> 0.9632 <SEP> 0.7594
<tb> 3 <SEP> 3 <SEP> 1.0000 <SEP> 0.9949 <SEP> 0.7560
<tb> 4 <SEP> 1.0000 <SEP> 0.7566
<tb> 5 <SEP> 1.0000
<Tb>
The homology between the two French strains Imp 1011-48121 and Imp 1 011-48285 is greater than 99% (0.9977).

 The homology between the two North American strains Imp 999 and Imp 1010 is also greater than 99% (0.9949). The homology between French strains and North American strains is a little over 96%.

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

 It is deduced that the strains according to 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 called porcine circovirus type II, PK / 15 representing type I. The strains belonging to this type II have a remarkable homogeneity of nucleotide sequence, even though they have isolated in very remote geographic areas.

Example 13 Analysis of Protein Encoded by the Genome of New Strains
VCP.

The nucleotide sequence of the lump isolate. 1010 was considered representative of other circovirus strains associated with generalized wasting syndrome. This sequence was further analyzed 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). It has been possible to detect 13 open reading frames (or COLs) larger than 20 amino acids in this sequence (circular genome). These 13 COLs are as follows

<tb> Name <SEP> Start <SEP> End <SEP> Strand <SEP> Size <SEP> of <SEP> COL <SEP> Size <SEP> Protein
<tb><SEP> (nucleotides <SEP> (nt)) <SEP>(SEP> amino acids <SEP> (aa))
<tb> COL1 <SEP> 103 <SEP> 210 <SEP> Meaning <SEP> 108 <SEP> nt <SEP> 35 <SEP> yy
<tb> COL2 <SEP> 1180 <SEP> 1314 <SEP> Meaning <SEP> 138 <SEP> nt <SEP> 45 <SEP> yy
<tb> COL3 <SEP> 1363 <SEP> 1523 <SEP> Meaning <SEP> 162 <SEP> nt <SEP> 53 <SEP> yy
<tb> COL4 <SEP> 398 <SE> 1342 <SEP> Meaning <SEP> 945 <SEP> nt <SEP> 314 <SEP> yy
<tb> COL5 <SEP> 900 <SEP> 1079 <SEP> Meaning <SEP> 180 <SEP> nt <SEP> 59 <SEP> yy
<tb> COL6 <SEP> 1254 <SEP> 1334 <SEP> Meaning <SEP> 81 <SEP> nt <SEP> 26 <SEP> yy
<tb> COL7 <SEP> 1018 <SEP> 704 <SEP> antisense <SEP> 315 <SEP> nt <SEP> 104 <SEP> yy
<tb> COL8 <SEP> 439 <SEP> 311 <SEP> antisense <SEP> 129 <SEP> nt <SEP> 42 <SEP> yy
<tb> COL9 <SEP> 190 <SEP> 101 <SEP> antisense <SEP> 90 <SEP> nt <SEP> 29 <SEP> yy
<tb> COL10 <SEP> 912 <SEP> 733 <SEP> antisense <SEP> 180 <SEP> nt <SEP> 59 <SEP> yy
<tb> COL11 <SEP> 645 <SEP> 565 <SEP> antisense <SEP> 81 <SEP> nt <SEP> 26 <SEP> yy
<tb> COL12 <SEP> 1100 <SEP> 1035 <SEP> antisense <SEP> 66 <SEP> nt <SEP> 21
<tb> COL13 <SEP> 314 <SEP> 1381 <SEP> antisense <SEP> 702 <SEP> nt <SEP> 213 <SEP> yy
<Tb>
The start and end positions of each COL refer to the sequence shown in FIG. 4 (SEQ ID No. 4). Of these 13 COLs, 4 show significant homology with analogous COLs located on the genome of the PCV PK-15 cloned virus. Each of the open reading frames present on the genome of all circovirus isolates associated with generalized wasting syndrome was analyzed. These 4 COLs are as follows

<tb> Name <SEP> Start <SEP> End <SEP> Strand <SEP> Size <SEP> of <SEP> Size <SEP> Protein <SEP> Mass
<tb><SEP> COL <SEP><SEP> (amino acids <SEP>) <SEP> molecular
<tb> COL4 <SEP> 398 <SE> 1342 <SEP> sense <SEP> 945 <SEP> nt <SEP> 314 <SEP> yy <SEP> 37.7 <SEP> kDa
<tb> COL7 <SEP> 1018 <SEP> 704 <SEP> antisense <SEP> 315nt <SEP> 104 <SEP> 11.8 <SEP> kDa
<tb> COL10 <SEP> 912 <SEP> 733 <SEP> antisense <SEP> 180 <SEP> nt <SEP> 59 <SEP> yy <SEP> 6.5 <SEP> kDa
<tb> COL13 <SEP> 314 <SE> 1381 <SEP> antisense <SEP> 702 <SEP> nt <SEP> 233 <SEP> aa <SEP> 27.8 <SEP> kDa
<Tb>
The start and end positions of each COL refer to the sequence shown in FIG. 4 (SEQ ID No. 4). COL size (in nucleotides = nt) includes the stop codon.

The comparison between the genomic organization of the PCV Imp. 1010 and PCV
PK-15 allowed the identification of 4 COLs conserved in the genome of both viruses. The table below shows the degrees of homology observed:

<tb> COL <SEP> lmp.1O10! COL <SEP> PCV <SEP> PK-15 <SEP> Percentage <SEP> of homology
<tb><SEP> COL4 / COL1 <SEP> 86 <SEP>%
<tb><SEP> COL13 / COL2 <SEP> 66.4 <SEP>%
<tb><SEP> COL7 / COL3 <SEP> 61.5 <SEP>% <SEP> (at the <SEP><SEP> level of the
<tb><SEP> recovery <SEP> (104 <SEP> aa)
<tb><SEP> COL10 / COL4 <SEP> 83 <SEP>% <SEP> (at the <SEP><SEP> level of the
<tb><SEP> recovery <SEP> (59 <SEP> aa)
<Tb>
The largest sequence identity was observed between the COL4 Imp. 1010 and COL1 PK-15 (86% homology). This was expected since this protein is probably involved in the replication of the viral DNA and is essential for viral replication (Meehan et al., J. Gen. Virol.1997, 78. 221227; Mankertz et al., J. Gen. Virol 1998.79.381-384).

The sequence identity between the COL13 Imp. 1010 and COL2 PK-15 is less strong (66.4% homology), but each of these two COLs does have a very conserved N-terminal basic region, which is identical to the N-terminal region of the structural protein. major CAV avian circovirus (Meehan et al., Arch.

Virol. 1992, 124. 301-319). Larger differences are observed between
COL7 Imp. 1010 and COL3 PK-15 and between COLI O lmp. 1010 and COL4 PK-15. In each case, there is a deletion of the C-terminal region of the COL7 and COL10 of the Imp isolate. 1010 when compared to COL3 and COL4 of PCV PK-15. The highest sequence homology is observed at the N-terminal regions of COL7 / COL3 (61.5% overlap homology) and
COL10 / COL4 (83% overlap homology).

 It appears that the genomic organization of the porcine circovirus is rather complex due to the extreme compactness of its genome. The major structural protein is probably derived from splicing between several reading frames located on the same strand of the porcine circovirus genome. It can therefore 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 usable antigen for specific diagnosis and / or vaccination. The invention therefore relates to any protein comprising at least one of these COLs. Preferably, the invention relates to a protein consisting essentially of COL4, COL7, COL10 or COL13.

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

 Plasmid pGEM-7/8 containing the complete genome (replicative form) of the lmp.999 isolate was transfected into PK / 15 cells according to the technique described by Meehan B. et al. Characterization of viral DNAs from cells infected with anemia: sequence analysis of cloned replicative form and transfection capabilities of cloned genome fragments, Arch Virol, 1992. 124.

301-319). The immunofluorescence analysis (see Example 4) carried out on the first passage after transfection on PK / 15 non-contaminated cells showed that the plasmid of clone pGEM7 / 8 was capable of inducing virus production.
Infectious PCV. The availability of a clone containing infectious PCV genetic material allows any useful manipulation on the viral genome to produce modified PCV viruses (either attenuated in pigs or defective) for the production of attenuated or recombinant vaccines, or for production of antigens for diagnostic kits.

Example 15 Production of PCV Antigens by In Vitro Culture
The culture of the PK / 15 non-contaminated cells and the viral multiplication are carried out according to the same modalities as in Example 1. The infected cells are harvested after trypsination after 4 days of incubation at 37 ° C. and digitized. The next passage is inoculated with 400,000 infected cells per ml.

Example 16: Inactivation of viral antigens
At the end of the viral culture, the infected cells are harvested and lysed by ultrasound (Branson Sonifier) or using a colloid mill rotor-stator (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 + 370C or with 0.5% beta-propiolactone for 24 hours at
+ 280C. If the virus titre before inactivation is insufficient, the virus suspension is concentrated by ultrafiltration using a membrane with a cut-off of 300 kDa (Millipore PTMK300). The inactivated virus suspension is stored at + 50C.

Example 17: Preparation of the vaccine as an emulsion based on mineral oil.

The vaccine is prepared according to the following formula - inactivated porcine circovirus suspension: 250 ml - Montanides ISA 70 (SEPPIC): 750 ml
The aqueous phase and the oily phase are sterilized separately by filtration.

The emulsion is prepared by mixing and homogenizing the ingredients using a Silverson turbine emulsifier.

 One dose of vaccine contains about 107'5 DIT50. The volume of a vaccine dose is 0.5 ml for intradermal administration, and 2 ml for intramuscular administration.

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

The vaccine is prepared according to the following formula - suspension of inactivated porcine circovirus: 200 ml - Dehymuls HRE 7 (Henkel): 60 ml - Radia 7204 (Oleofina): 740 ml
The aqueous phase and the oily phase are sterilized separately by filtration. The emulsion is prepared by mixing and homogenizing the ingredients using a Silverson turbine emulsifier.

 One dose of vaccine contains about 107'5 DIT50. The volume of a vaccine dose is 2 ml for intramuscular administration.

EXAMPLE 19 Indirect Immunofluorescence Results Versus US and French PCV Virus Strains and PK / 15 Contaminant with Hyperimmune Serum (PCV-T), a Panel of F99 Monoclonal Antibodies, Prepared from PK And a hyperimmune serum prepared from the Canadian strain (PCV-C)
VIRUS

<tb><SEP> PK / 15 <SEP> USA <SEP> France
<tb> PCV-T <SEP> antiserum <SEP> 2 <SEP> 6 <SEP> 400 <SEP> 200 <SEP> 800
<tb> PCV-C <SEP> antiserum <SEP> 200 <SEP>#<SEP><SEP> 6,400 <SEP> 2 <SEP> 6,400
<tb> F99 <SEP> 1H4 <SEP> | <SEP>#<SEP> 10 <SEP> 000 <SEP> | <SEP><SEP><<SEP> 100 <SEP> | <SEP> 100
<tb> F99 <SEP> 4B10 <SEP> 210 <SEP> 000 <SEP><100<SEP><100
<tb> F99 <SEP> 2B7 <SEP> | <SEP>#<SEP><SEP> 10 <SEP> 000 <SEP> | <SEP> 100 <SEP> | <SEP><SEP> 100
<tb> F99 <SEP> 2E12 <SEP>#<SEP> 10 <SEP> 000 <SEP><SEP><100<SEP><100
<tb> F99 <SEP> 1C9 <SEP>#<SEP> 10 <SEP> 000 <SEP><SEP><100<SEP> 100
<tb> F99 <SEP> 2E1 <SEP> | <SEP>#<SEP> 10 <SEP> 000 <SEP> | <SEP><SEP><<SEP> 100 <SEP> | <SEP><SEP> 100
<tb> F99 <SEP> 1H4 <SEP>#<SEP> 10 <SEP> 000 <SEP><SEP> 100 <SEP><100
<tb> inverse of the last dilution of the serum or monoclonal antibody that gives a positive reaction by indirect immunofluorescence.

Claims (7)

 A polypeptide comprising at least one type II porcine circovirus COL, selected from the group of COLs 1 to 1 3.  2. Polypeptide according to claim 1, characterized in that it is chosen from COLs 4, 7, 10 and 13.  3. A subunit vaccine, comprising at least one polypeptide according to claim 1 or 2, in a veterinarily acceptable diluent or carrier, and optionally a veterinarily acceptable adjuvant.  An in vivo expression vector comprising, integrated in its genome, a DNA fragment expressing in vivo a polypeptide according to claim 1 or 2.  5. Expression vector according to claim 4, characterized in that it is selected from living viruses capable of multiplying in the pig without being pathogenic for this animal, and the plasmids.  6. Expression vector according to claim 5, characterized in that the viral vector is chosen from porcine herpes viruses, such as Aujeszky's disease virus, porcine adenovirus, poxviruses, in particular the virus of the invention. vaccinia, Avipox, canarypox, swinepox.  7. Live or plasmid vaccine, characterized in that it expresses a polypeptide according to claim 1 or 2.  The invention relates to novel strains of porcine circovirus isolated from pulmonary or glanglionic specimens from culture affected by the syndrome of generalized post-weaning dieback (in English PMWS).  ABSTRACT OF THE TECHNICAL CONTENT OF THE INVENTION  New porcine circoviruses. vaccines and diagnostic reagents  Merial. Queen's University of Belfast and University of Saskatchewan It relates to purified preparations of these strains, conventional attenuated or in vivo vaccines, recombinant live vaccines, plasmid vaccines and subunit vaccines, as well as reagents and diagnostic methods. It also relates to DNA fragments that can be used for the production of subunits in an in vitro expression vector or as sequences to be integrated into an in vivo expression vector of the virus or plasmid type.