Ii type porcine circovirus and uses thereof

FIELD: veterinary virology.

SUBSTANCE: invention relates to 5 strains of II type porcine circovirus (PCV II) which represents causative agent of porcine post-wealing multy-systemic wasting syndrome (PMWS). Disclosed are various immunogenic compositions and vaccine based on said strains for PMWS prophylaxis and/or treatment. Also disclosed are vectors, viral preparations, cell extracts, cell culture supernatants, containing PCV II or nucleotide or protein components thereof; method for PCV II diagnosis, as well as diagnostic composition and kit.

EFFECT: new agent for treatment of porcine PMWS.

178 cl, 7 dwg, 5 tbl, 19 ex

 

The present invention relates to new strains of swine circovirus (commonly referred to as PCV), responsible for PMWS syndrome (multiboot wasting syndrome pigs, also called a lot of system wasting syndrome after cessation of breastfeeding), reagents and methods to detect them, methods of vaccination and vaccines, as well as ways to obtain these reagents and vaccines.

PCV originally discovered as narutoporno contaminant in cell lines RK/15 kidney of pigs. This virus is classified as belonging to the Circoviridae, which also include the chicken anemia virus (commonly referred to as CAV) and the virus PBFDV (virus diseases of the beak and feathers of parrots). This small (15 to 24 nm), without shell viruses, a common characteristic of which is that they contain the genome in the form of a circular single-stranded DNA ranging in size from 1,76 up 2,31 TPN Initially assumed that this gene encodes a polypeptide with a mass of about 30 kDa (Todd et al., Arch. Virol. 1991, 117; 129-135). However, recent studies have shown a more complex transcription (Meehan B.M. et al., 1997, 78; 221-227). Moreover, it is not found significant homology in nucleotide sequences or normal antigenic determinants between the three known types of circoviruses.

PCV originating from cells of the RK/15, is considered non-pathogenic. It p is the sequence known from ..Meehan et al., J. Gen. Virol., 1997 (78) 221-227). More recently, some authors believed that the PCV strains can 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. Swine Prac. 1997; 499-501). Nayar et al. using the PCR method detected PCV DNA in pigs with PMWS syndrome. However, neither strain of PCV wild-type is still not selected.

The PMWS syndrome, found in Canada, in the United States of America and France, clinically characterized by progressive weight loss and such manifestations as tachypnea (rapid breathing without deepening it), shortness of breath, and jaundice. From the point of view of pathology it is manifested linfocitos or granulomatoses infiltration, lymphadenopathy and, rarely, hepatitis or impositionem or granulomatosis nephritis (E.G. Clark, Proc Am. Assoc. Swine Prac. 1997; 499-501; La critics ' Vétérinaire No. 26, supplement to La critics ' Vétérinaire 1996 (834); La critics ' Vétérinaire 1997 (857): 54; (Gupi P.S. Nayar et al., Can. Vet. J., vol.38, 1997: 385-387).

The applicant failed to allocate five new strains of PCV from lung and retinal samples obtained from farms located in Canada, in the United States (California) and France (Brittany), hereinafter called circovirus in accordance with the present invention. These viruses are found in places with pathological changes in pigs with PMWS syndrome and in healthy pigs such viruses are not detected.

In addition, the applicant has deciphered Geno is s four of these strains, namely strains obtained from Canada, USA, and two strains from France. These strains show a very strong homology with respect to each other at the nucleotide level in excess of 96%, and a much weaker relative to the strain RK/15 - about 76%. Thus, new strains can be considered as representatives of a new type of circoviruses pigs, which are here referred to as type II and type I presents RK/15.

Therefore, the object of the present invention are circovirus pigs type II, as defined above, isolated or purified form of the drug.

The invention applies to any circoviruses pigs, which can be distinguished from the physiological sample or tissue sample, especially from the affected areas, from sick pigs with PMWS syndrome, especially using the method described in the examples, in particular, circovirus type II.

More specifically, the object of the present invention are purified preparations of five strains deposited in ECASS (European collection of cell cultures, Centre for applied Microbiology and research, Porton Down, Salisbury, Wiltshire S54 OJG, United Kingdom)on Thursday, 2 October 1997:

- collection No. V97100219 (called here Imp.1008PCV);

- collection No. V97100218 (called here Imp.1010PCV);

- collection No. V97100217 (called here Imp.999PCV);

and received on Friday 16 anwara:

- collection No. V98011608 (called here Imp.1011-48285);

- collection No. V98011609 (called here Imp.1011-48121).

The invention aims to review circoviruses pigs, isolated from diseased pigs, and/or circoviruses with significant serological similarity with strains of the present invention, and/or circoviruses, cross-hybridization with strains of the present invention, in the conditions of such severity that they did not allow the implementation of hybridization with the strain of PCV RK/15.

Viral strains isolated from a physiological sample or tissue sample, especially from the affected area, from pigs with PMWS syndrome, can be successfully propagated on cell lines, such as cell line kidney of pigs, in particular cells of the RK/15, available from contamination (in particular, for the PCV, and pestiviruses, adenoviruses pigs and swine parvovirus), with a view to their reproduction and especially for the production of their antigen, whole (e.g., virus) and/or its subunits (e.g., polypeptides).

Very interestingly and unexpectedly, that these isolates proved to be very productive in culture cells RK/15, which has the obvious advantage for the production of virus or antigen, in particular for the production of inactivated vaccines.

The subject of this is part II of the invention are also drugs circoviruses, isolated after passage the cells, especially cell lines, such as the cells of the RK/15 in vitro when investirovanie at least one circoviruses of the present invention or any circovirus pigs, which can be isolated from a physiological sample or tissue sample, especially from the affected areas, from pigs with PMWS syndrome. The aim of the invention is also a culture extract or supernatant, which can be purified using standard methods, and in General any antigenic preparations obtained from cultures in vitro.

The subject invention are also immunogene-active ingredients and vaccines containing at least one antigen as defined above.

They may represent immunogene-active ingredients, based on attenuating (weakened) live whole virus or vaccine, obtained with the help of these active ingredients, and attenyerevan (weakening) carried out by conventional means, for example, by passage to the cells, preferably by passage to the cells of the pig, especially on the lines, such as cell line RK/15 (using, for example, from 50 to 150, especially about 100 passages). These vaccines typically include a carrier or diluent suitable with the veterinary point of view of the Oia, optional adjuvant suitable with the veterinary point of view, and optional stabilizer for lyophilization.

These vaccines are preferably contain from 103up to 106TCID50.

They may represent immunogene-active ingredients or vaccines based on circovirus antigen in accordance with the present invention, in inaktivirovannoj condition. In addition, the vaccine includes a carrier or diluent suitable with the veterinary point of view, with optional addition of adjuvant suitable with the veterinary point of view.

Circovirus of the present invention with factions that may be present, inactivate, in accordance with methods known to experts in this field of technology. Inactivation is preferably carried out chemically by, for example, by treatment of the antigen with a chemical agent, such as formaldehyde (formalin), paraformaldehyde, β-propiolactone or ethylenimine or its derivatives. The preferred method of inactivation for this invention is the treatment with a chemical agent, in particular ethylenimine or β-propiolactone.

Preferably, the inactivated vaccine of the present invention add an adjuvant, which is best used in the form of emulsions such as emulsions of water in oil or oil in water, the CE is provided with ways, well-known specialists in this field of technology. It is also possible to adjuvant came from conventional adjuvant compounds, the active ingredient.

Of adjuvants that can be used, as examples can be mentioned aluminum hydroxide, saponin (e.g., saponin Typically or Quil A; see Vaccine Design, The Subunit and Adjuvant Approach, 1995, edited by Michael F. Powell and Mark J. Newman, Plennum Press, New York and London, p.210), Avridine®(Vaccine Design, p.148), DDA (bromide dimethyldioctadecyl-ammonium, Vaccine Design, p.157), polyphosphazene (Vaccine Design, p.204) or alternatively, emulsion oil in water, based on mineral oil, squalene (e.g., emulsion SPT, Vaccine Design, p.147), squalene (e.g., MF59, Vaccine Design, p.183), or emulsions of water in oil, based on metabolisable oil (preferably in accordance with the publication WO-A-94 20071), as well as emulsions, described in US-A-5422109. You can also choose a combination of adjuvants, for example Avridine®or DDA, with the emulsion.

These vaccines are preferably contain from 106up to 108TCID50.

Adjuvants for live vaccines can be selected from adjuvants provided for inactivated vaccines. The preferred emulsion. To adjuvants specified for inactivated vaccines, you can add adjuvants described in the publication WO-A-9416681.

As a stabilizer for lyophilization, can be mentioned, for 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 phosphates of alkali metals.

In addition, the applicant received the genomes of four isolates designated as sequence (CONSECUTIVE.) No. 1 to 4 and optionally No. 6.

Therefore, the object of the present invention is a DNA fragment containing all of the sequences or part thereof. Needless to say, that the invention automatically covers all equivalent sequences, such as sequences that do not alter the functional properties or the strain-specificity of the described sequence or encoded by this sequence of polypeptides. Of course, the invention also include sequences that differ due to the degeneracy of the code.

The invention also encompasses sequences that are equivalent in the sense that they are able to gibridizatsiya with the above-described sequence under conditions of high severity and/or have a high homology with the strains of the present invention and belong to the group II, as defined above.

These sequences and their fragments can be conveniently used for the expression of polypeptides in vitro or in vivo using the corresponding age of the Directors.

In particular, open reading frames that make up the DNA fragments in accordance with the present invention, which can be used to obtain this effect, identified in the genomic sequence circovirus type II. The invention relates to any polypeptide containing at least one of these open reading frames (corresponding amino acid sequence). Preferably, the invention relates to protein, essentially consisting of ORF4, ORF7, ORF10 or ORF13 (ORF - open reading frame).

For the expression of subunits in vitro as a means of expression, preferably using E. coli or baculovirus (US-A-4745051). The coding sequence (or sequences) or fragments thereof is inserted into the genome of the baculovirus (filamentous virus (e.g. baculovirus Autographa californica Nuclear Polyhedrosis Virus AcNPV), and then the last propagated in insect cells such as Spodoptera frugiperda Sf9 (Sample of American cell culture collection of ADS CRL 1711). Subunit can also be produced in eukaryotic cells such as yeast (e.g., Saccharomyces cerevisiae) or in mammalian cells (e.g. cells Cho - Chinese hamster ovary, cells KSS).

The object of the present invention are polypeptides produced in vitro using these tools, expression, and then optional cleansing is presented in accordance with conventional methods. The subject invention is also a subunit (based on subunits) vaccine, comprising at least one thus obtained polypeptide or fragment, in a carrier or diluent suitable with the veterinary point of view, and optional adjuvant suitable with the veterinary point of view.

For expression in vivo, for the production of recombinant live vaccines, the coding sequence or sequences or fragments thereof is inserted into a suitable expression vector under conditions that ensure the expression of this polypeptide or polypeptides. Suitable vectors can be used live viruses, preferably capable of reproduction in pigs, non-pathogenic for pigs (with natural or artificially attached to hepatogenous), in accordance with methods well-known to specialists in this field of technology. In particular, you can use the herpes viruses of pigs, such as the virus of Aujeszky's disease (false rabies), adenovirus pigs, the viruses of smallpox, especially vaccinia virus, avian pox, smallpox Canaries, the smallpox virus of pigs. As vectors can also be used plasmid DNA (WO-A-9011092, WO-A-9319813, WO-A-9421797, WO-A-9520660).

The object of the present invention therefore also are vectors and recombinant live vaccines or plasmide the vaccine (polynucleotide or DNA vaccines), thus obtained, and these vaccines include, in addition, a carrier or diluent suitable with the veterinary point of view.

Vaccines in accordance with the present invention (live attenuated, inactivated, subunit, recombinant and plasmid vaccine) may include one or more active ingredients (antigens) one or more (2 or 3) circoviruses in accordance with the present invention.

For each of the above types of vaccine the present invention also provides a combination vaccine circoviruses pigs by vaccination against other pathogens of pigs, in particular against those that may be associated with the syndrome PMSW. Therefore, vaccines in accordance with the present invention, in particular inactivated, can enable a different valency corresponding to another pathogen of pigs. Among these other pathogens of pigs, preferably can be called PRRS (reproductive and respiratory syndrome swine) (specialists in the art can refer to the following publications: WO-A-93/07898, WO-A-94/18311, FR-A-2709966; .Chareyre et al., Proceedings of the 15thIPVS Congress, Birmingham, England. 5-9 July 1998, p.139, which are included in the present description as a reference) and/or Mycoplasma hyopneumonia (specialists in the art can refer to the following publications: EP-A-597852, EP-A-550477, EP-A-571-648; .Matinon et al., R, 284, 285 and G.Reynaud et al., p.150, all in wyrezyserowany edition of the Proceedings of the 15thIPVS Congress, which are included in the present description as references). Among other interesting valences include Actinobacillus pleuropneumoniae, E. coli, atrophic rhinitis of pigs, as well as false rabies (Aujeszky's disease), cholera swine swine flu.

The object of the present invention is also a way to induce the pigs immune response to circovirus in accordance with the present invention. The subject of the invention, in particular, is a method of vaccination, which is effective for pigs.

The method proposes the introduction of pigs, one or more portions of the above vaccines. You can also combine several of the above vaccines in the same scheme of vaccination.

This method is applicable not only for adult pigs, but also for pigs or for pregnant females. Vaccination last makes possible the creation of passive immunity in the newborn (antibodies of maternal origin).

The invention also provides a diagnostic of the presence of circoviruses of the present invention in pigs. The subject of the invention therefore are also diagnostic tests and methods of diagnosis associated with the use of reagents that will be described below.

Knowledge of the sequence is different circoviruses allows you to determine the overall sequence, which in turn makes it possible for reagents that can detect all known circovirus pigs.

Specialists in the art can also select fragments of the sequences corresponding to the fields, only to a small extent exhibiting homology or not show homology with the corresponding sequence of circovirus RK/15, in order to supply a specific diagnosis.

Comparison of sequences allows specialists in the art to select a reagent in accordance with their wishes.

The first reagent described here consists of DNA sequences and their fragments, which, in particular, can be used as probes or primers in the well-known methods, hybridization or PCR (polymerase chain reaction).

The second reagent consists of polypeptides encoded by those sequences of the virus, or expressed using vector (see above), or chemically synthesized in accordance with conventional methods of peptide synthesis.

The third and fourth reagents respectively consist of polyclonal and monoclonal antibodies, which can be obtained in accordance with customary methods viruses, polypeptides, or fragments, extracted or encoded by DNA sequences.

These second third and fourth reagents can be used in the diagnostic method, one of the objects of the present invention, which are tested on a sample of physiological fluid (blood, plasma, serum and the like) or a sample of tissue (ganglia, liver, lungs, kidneys and the like)obtained from the subject for testing pigs for the presence of antigen-specific circovirus in accordance with the present invention, by searching for and detecting either the antigen or antibodies directed against this antigen.

The antigens and antibodies of the present invention can be used in any known laboratory diagnostic methods.

However, it is preferable to use them in methods that can be used directly in the field veterinarian, breeder or pet owner. Specialists in this field technicians have at their disposal a variety of laboratory and field methods, and so it can adapt the use of this antigen and/or antibody as a reagent or reagents for diagnostics.

Diagnostic methods should preferably be used in the framework of the present invention are Western blotting, immunofluorescence assay, ELISA (enzyme-linked immunosorbent assay) and immunochromatography.

With regard to the use of chromatography methods, specialists can refer, in particular, to the publ the requirements Robert F.Zurk et al., Clin. Chem. 31/7, 1144-1150 (1985), as well as to patents and patent applications WO-A-88/08 534, WO-A-91/12528, EP-A-291176, EP-A-299428, EP-A-291194, EP-A-284232, US-A-5120643, US-A-5030558, US-A-5266497, US-A-4740468, US-A-5266497, US-A-4855240, US-A-5451-504, US-A-5141850, US-A-5232835 and US-A-5238652.

Accordingly, it is preferable to carry out the search and detection of specific antibodies in the sample using an indirect test using competition or substitution. To do this, as a reagent for the diagnosis using the antigen or a fragment of this antigen, can still be recognized by antibodies. Tagging can be successfully implemented by tagging peroxidase or with the help of special methods of tagging, preferably colloidal gold.

It may also be desirable detection of the antigen in the sample using labeled antibodies specific for a particular antigen. Tagging is best done as described above.

Under an antibody specific to the antigen that can be used, in particular, when competition or substitution of or for the detection of the antigen refers to monoclonal or polyclonal antibodies specific to the antigen, fragments of these antibodies, preferably Fab fragments or F(ab)'2.

Another symptom of this invention to provide a polyclonal or monoclonal antibodies specific for the antigen in accordance with the present and the acquisition, moreover, these antibodies can then be used, in particular, as reagents for the diagnosis, detection of antigen in a sample of physiological fluid or tissue sample, or even to detect antibodies present in these samples. The invention also includes immunologically functional fragments of these antibodies, in particular the fragments F(ab) or F(ab)'2.

Antibodies can be obtained in the usual way. It is possible, in particular, refer to the following publications: Antibodies, A Laboratory Manual, 1988, Cold Spring Harbor Laboratory, USA or J.M.Goding, Monoclonal Antibodies: Principles and Practice, Academic Press Inc., the contents of which are incorporated into this description by reference.

In particular, it is possible, as is known per se (in itself), to carry out the fusion of spleen cells of mice immunized with the antigen or at least one of its fragments, corresponding myelomatose cells.

The object of the present invention is also preferably receive treated or partially treated or even untreated monoclonal or polyclonal antibodies specific for the antigen, especially mouse or rabbit antibodies.

The present invention also allows you to determine interest epitopes, especially on the basis of these DNA sequences, regardless of whether these epitopes interest on what I vaccines or diagnostics. Based on the DNA sequence of the genome of circovirus in accordance with the present invention, the specialists in the art is able to determine the epitopes in accordance with known methods, for example using an appropriate computer program or method PEPSCAN. Epitopes are immunodominant region of protein located on the surface of proteins. Therefore, they can detect antibodies and therefore they are particularly applicable in the field of diagnostics or to generate antibodies for diagnostic purposes or for the production of the corresponding peptides that can be used as reagents for the diagnosis.

At least, the epitope is a peptide having from 8 to 9 amino acids. Usually, it is preferable that the epitope had minimum 13 to 25 amino acids.

Therefore, experts in the art is able, using one or more of these methods and other suitable methods, to find the epitopes for use peptides or antibodies for diagnostic purposes.

The object of the present invention is also a kit for the diagnosis, including the antigen and/or polyclonal or monoclonal antibodies specific for this antigen. In particular, it sets for the diagnosis corresponding to the above-described diagnostic methods.

Gleasonian described in more detail, using permissive, are given as examples of variants of its implementation with reference to the drawings, 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.

Figure 5: Comparison of 4 sequences in accordance with figure 1-4 sequence strain of PCV RK/15.

6: DNA Sequence of the genome of strain Imp.999 defined when it is first registered in France on 3 October 1997.

Fig.7: comparison of the sequence 6 sequence of strain RK/15.

The list of sequences No. of sequences.

PLACENTAS. No. 1: DNA Sequence of the genome of strain Imp. 1011-48121.

PLACENTAS. No. 2: DNA Sequence of the genome of strain Imp. 1011-48285.

PLACENTAS. No. 3: DNA Sequence of the genome of strain Imp. 999.

PLACENTAS. No. 4: the DNA Sequence of the genome of strain Imp. 1010.

PLACENTAS. No. 5: the DNA Sequence of the genome of strain RK/15.

PLACENTAS. No. 6: the DNA Sequence of the genome of strain Imp. 999 defined when it is first registered in France on 3 October 1997.

EXAMPLES

EXAMPLE 1. Cultivation and isolation of strains circoviruses pigs.

Tissue samples were taken in France, in Canada and in the United States from the lungs and lim is oticheskih nodes of pigs. These pigs showed clinical signs typical multiboot wasting syndrome after cessation of breastfeeding. To facilitate virus isolation, tissue samples immediately after autopsy (necropsy) were frozen at -70°.

To highlight viruses were prepared slurry containing about 15% of the tissue sample, containing salt minimal medium by Earl (EMEM, BioWhittaker UK Ltd., Wokingham, UK), penicillin (100 μg/ml) and streptomycin (100 μg/ml) (Wednesday MEM-SA), by grinding the tissue with sterile sand using a mortar and pestle. Crushed the drug is tolerated on Wednesday MEM-SA, and then centrifuged at 3000 g for 30 minutes at +4°to receive and collect the supernatant.

Before inoculation of cell cultures to 2 ml of each supernatant was added 100 μl of chloroform and was continuously stirred for 10 minutes at room temperature. This mixture then was transferred into a microcentrifuge tube, centrifuged at 3000 g for 30 minutes, then collected the supernatant. The supernatant was used as inoculum for the experiments on the selection of viruses.

All studies on virus isolation was performed on cell cultures RK/15 for whom it was known that they are not infected with swine circovirus (PCV), pestiviruses, adenovirus pigs and Pervov the Russ pigs (Allan G. et al., Pathogenesis of porcine circovirus experimental infections of colostrum-deprived piglets and examination of pig foetal material. Vet. Environ. 1995, 44, 49-64).

The selection of circoviruses pigs was carried out in accordance with the following method:

Monolayers of cells RK/15 dissociatively by trypsinization (a mixture of trypsin and versene) from confluent (merging) of the cultures, and then transferred on Wednesday MEM-SA containing 15% fetal calf serum, not contaminated by pestiviruses (Wednesday MEM-G)with a final concentration of about 400,000 cells per 1 ml Fractions of this suspension by aliquot of 10 ml was mixed with 2 ml of the aliquot fractions of the above inoculum and the final mixture was transferred by aliquot 6 ml 2 Falkovsky bottle 25 cm2. Then these cultures were incubated at a temperature of +37°C for 18 hours in an atmosphere of CO2.

After incubation, the culture medium polyethlyene the monolayers were treated with 300 mm D-glucosamine (Cat. No. G48175, Sigma-Aldrich Company Limited, Poole, UK) (Tischr I. et al., Arch. Virol., 1987, 96, 39-57), then continued incubation for 48-72 hours at +37°C. After this final incubation, one of the two Falkovsky vials for each inoculum was subjected to three consecutive cycles of freezing/thawing. Cells RK/15 of the remaining vials were treated with a solution of a mixture of trypsin and versene, re-suspended in 20 ml of medium MEM-G, and then inocular the Wali in valkanovska bottles 75 cm 2at a concentration of 400,000 cells/ml Then sizeincrement vials were superintegrable by adding 5 ml of the appropriate lysate obtained after cycles of freezing/thawing.

EXAMPLE 2. Obtaining samples of cell cultures for the detection of circoviruses pigs using immunofluorescence assay or by in situ hybridization.

Selected 5 ml superinfections suspension and inoculable in Petri dishes with a diameter of 55 mm, which were sterile skimmed glass cover. Culture in bottles and on cover glasses were incubated at +37°and were treated with glucosamine as described in example 1. After 24 to 48 hours after treatment with glucosamine culture on cover glasses were fixed either with acetone for 10 minutes at room temperature, or 10%buffered formaldehyde for 4 hours. After this commit all to use fixed glasses for research on in situ hybridization and immunocytochemical methods cover glasses were stored at -70°With on silica gel.

EXAMPLE 3. Detection methods sequences PCV by in situ hybridization.

The in situ hybridization was performed on tissues taken from sick pigs and fixed with formaldehyde, as well as on the preparations of cell cultures inoculated to highlight viruses (see example 2) and fixed is the R on top of the glasses.

Used complete genomic probes corresponding to circoviruses pigs (PCV) PK/15 and infectious chicken anemia virus (CAV). Plasmid pPCV1 containing the replicative form of the PCV genome, cloning in the form of a single insert size 1.7 thousand base pairs (KBP) (Meehan C. et al. Sequence of porcine circovirus DNA: affinities with plant circoviruses, J. Gen. Virol. 1997, 78, 221-227), used as a source of virus-specific PCV DNA. A similar plasmid, rsaa containing the replicative form of circovirus birds CAV size 2.3 KBP, was used as negative control. Appropriate glycerole stocks of these two plasmids were used for production and purification of plasmids by the method of alkaline lysis (Sambrook J. et al. Molecular cloning: A laboratory Manual, 2ndEdition, Cold Spring Harbor laboratory, Cold Spring Harbor, New York, 1989) in order to continue to use them as matrices to obtain probes. Circoviruses probes, representative for the complete genome of PCV and CAV, obtained from purified plasmids described above (1 µg for each probe), and from hexanucleotide primers, which randomly used a commercial kit, non-radioactive labels ("DIG DNA labeling kit, Boehringer Mannheim, Lewes, UK)according to the manufacturer's recommendations.

Tagged digoxigenin probes were dissolved in 50-100 ál of sterile water before use for in situ hybridization.

Tissue samples Bo is lnyh pigs, enclosed in paraffin and fixed with formaldehyde and drugs living cell cultures, fixed with formaldehyde, obtained for the detection of nucleic acids PCV in accordance with the following method:

From tissue blocks embedded in the wax, making slices with a thickness of 5 μm, liberated them from the paraffin, and then degidrirovanie in solutions with successively decreasing concentrations of alcohol. The tissue sections and cell cultures, fixed with formaldehyde, incubated respectively for 15 and 5 minutes at +37°in a solution of 0.5% proteinase K in 0.05 M Tris-HCl buffer containing 5 mm EDTA (pH 7,6). Then the slide with the preparations were placed in a 1% solution of glycine in autoclaved distilled water for 30 seconds, washed twice in 0.01 M PBS (saline phosphate buffer) (pH of 7.2), and finally washed for 5 minutes in sterile distilled water. Then finally dried in the open air and brought into contact with the probes.

Each preparation of tissue/probe was covered with pure skimmed cover glass, and then placed in an oven with a temperature of +90°10 minutes, put a piece of ice for 1 minute, and finally incubated for 18 hours at +37°C. Then the drugs briefly immersed in 2XSSC (pH 7.0)to remove the protective glass cover, and then twice for 5 min were washed in 2X SSC and finally, twice for 5 minutes and washed with PBS.

After these washes, the sections 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 1% solution of blocking reagent (Cat. No. 1096176, Boehringer Mannheim, Lewes, East Sussex, UK) in maleic buffer for 20 minutes at +37°C.

Then the preparations were incubated with 1/250 solution antidigoxigenin monoclonal antibodies (Boehringer Mannheim) in blocking buffer for 1 hour at +37°C, washed with PBS and finally incubated with biotinylating artemisinine antibodies for 30 minutes at +37°C. the Preparations were washed in PBS, the activity of endogenous peroxidase was blocked by treatment with 0.5% hydrogen peroxide solution in PBS for 20 minutes at room temperature. Then the preparations were again washed in PBS and incubated with the substrate 3-amino-9-diethylcarbamoyl (AEC) (Cambridge Bioscience, Cambridge, UK), prepared immediately prior to use.

After final washing with tap water samples were subjected to contrast staining with hematoxylin, "polsinelli" under tap water, and then poured on top of the glass for microscope priming fluid (GVA Mount, Cambridge Bioscience, Cambridge, UK). Control options include the use of inappropriate negative probe (CAV) and the positive probe (PCV) on the sample is x, obtained from diseased pigs and from healthy pigs.

EXAMPLE 4. Method of detection of PCV using immunofluorescence assay

Initial screening of all preparations of cell cultures, fixed with acetone was carried out by the method of indirect immunofluorescence assay (IIF) using a pool of sera of adult pigs in a dilution of 1/100. This pool of sera included sera from 25 adult pigs from Northern Ireland, and also contained antibodies against a wide range of viruses of pigs, including PCV, swine parvovirus, adenovirus pigs and the PRRS virus (reproductive and respiratory syndrome swine). The analysis method IIF was performed by incubation of serum (diluted PBS) with cell cultures for 1 hour at +37°With two subsequent washing PBS. Then the cell culture is incubated with diluted 1/80 PBS with rabbit antibodies to swine immunoglobulin conjugated with isothiocyanato fluorescein (FITC) for 1 h, then washed with PBS and embedded in glycerol buffer before microscopic examination under ultraviolet light.

EXAMPLE 5. The results of in situ hybridization in tissues of patients pigs.

In situ hybridization carried out using the probe on the basis of the PCV genome isolated from tissues taken from the French, canadian and Californian piglets with pathological changes, calling the installed multiboot wasting syndrome, and fixed with formaldehyde, demonstrated the presence of nucleic acids PCV related to the affected areas, in several of the studied affected areas. No signals were observed when the probes on the basis of the genome of PCV used on tissue taken from healthy pigs, or when the tissues of patients pigs used probe CAV. The presence of nucleic acid PCV identified in the cytoplasm and nucleus of numerous mononuclear cells, infiltra diseased areas in the lungs of California piglets. It was also demonstrated the presence of nucleic acid PCV in pneumocytes, bronchial and bronchiolar epithelial cells, and endothelial cells of arterioles, venules and lymphatic vessels.

Patients piglets from France the presence of nucleic acid PCV found in the cytoplasm of numerous follicular lymphocytes and intraseasonally mononuclear cells of lymph nodes. Nucleic acid PCV also found in some cincytech. Depending on the results of detection of nucleic acid PCV, samples of lung California swine lymph nodes of the mesentery French pigs and bodies of canadian pigs used for the selection of new strains of circoviruses pigs.

EXAMPLE 6. The results of kultivirovaniya with new strains of circovirus in pigs and detection by immunofluorescence assay

No cytopathic effect was not observed in cell cultures inoculated with samples taken from the French pigs (strain Imp.1008), California pigs (strain Imp.999) and the canadian pigs (strain Imp.1010), showing clinical signs of the multiboot wasting syndrome. However, immune staining of drugs derived from inoculated cell cultures, fixed with acetone, and a pool of pig polyclonal sera, showed nuclear fluorescence in numerous cells in cultures inoculated with the use of light California pigs (strain Imp.999), using the lymph nodes of the mesentery French pigs (strain Imp.1008) and using the bodies of canadian pigs (strain Imp.1010).

EXAMPLE 7. Extraction of genomic DNA circoviruses pigs.

Replicative form new strains of swine circovirus (PCV) was obtained by infetsirovaniya cell cultures RK/15 (see example 1) (10 vials of 75 cm3)collected after 72-76 hours of incubation and treated with glucosamine as described for cloning of replicative forms CAV (Todd D. et al. Dot blot hybridization assay for chicken anemia agent using a cloned DNA probe. J. Clin. Environ. 1991, 29, 933-939). Double-stranded DNA replicative forms were extracted in accordance with a modification of the method of Hirt (Hirt C. Selective extraction of polyoma virus DNA from infected cell cultures, J. Mol. Biol., 1967, 36, 365-369), described what Molitor (T.W. Molitor et al. Porcine parvovirus DNA: characterization of the genomic and replicative form DNA of two virus isolates, Karpova et al., 1984, 137, 241-254).

EXAMPLE 8. Restriction map of the replicative form of the genome of strain Imp.999 of circovirus pigs.

DNA (1-5 μg), extracted using the method of hirta, was treated with S1-nuclease (Amersham) in accordance with the recommendations of the provider, and then this DNA was digested various restriction enzymes (Boehringer Mannheim, Lewes, East Sussex, UK) and the cleavage products were separated by electrophoresis in 1.5%agarose gel in the presence of ethidium bromide, as described Todd and others (Purification and biochemical characterization of chicken anemia agent. J. Gen. Virol. 1990, 71, 819-823). DNA extracted from cultures of strain Imp.999, has a unique EcoRI site, the two sites SacI and has no site > PST. Therefore, this profile restriction differs from the profile restriction strain PCV PK/15 (Meehan C. et al. Sequence of porcine circovirus DNA; affinities with plant circoviruses, 1997, 78; 221-227), which, conversely, has a website > PST and does not have a single EcoRI site.

EXAMPLE 9. Cloning of the genome of strain Imp.999 of circovirus pigs.

Restriction fragment size of about 1.8 KBP obtained by cleavage of double-stranded replicative form of the strain of PCV Imp.999 EcoRI, was isolated after separation by electrophoresis in 1.5%agarose gel (see example 3) using a commercial kit Qiagen (QIAEXII Gel Extraction Kit, Cat # 20021, QIAGEN Ltd., Crawley, West Sussex, UK). The fragment EcoRI-EcoRI then luigirules vector pGEM-7 (Promega, Medical Supply Company, Dublin, Ireland), previously cleaved with the same restriction enzyme and dephosphorylated in accordance with standard cloning methods (Sambrook J. et al., Molecular cloning: A laboratory Manual, 2ndEdition, Cold Spring Harbor laboratory, Cold Spring Harbor, New York, 1989). The obtained plasmids were used to transform the Escherichia coli strain JM109 (Stratagene, La Jolla, USA) according to standard methods. Restriction fragment EcoRI-EcoRI strain of PCV Imp.999 also cloned in the EcoRI site of the vector pBlueScript SK+ (Stratagene Inc., La Jolla, USA). Among the clones obtained from each strain, took at least 2 clones containing fragments of the expected size. The resulting clones were then cultured in a small volume (2 ml) or in a large volume (250 ml) and was isolated plasmid containing the complete genome of strain Imp.999, in accordance with the standard methods of obtaining and purification of plasmids.

EXAMPLE 10. Sequencing of genomic DNA (double-stranded replicative form) strain PCV Imp.999.

The nucleotide sequence of the two EcoRI clones (clone pGEM-7/2 and pGEM-7/8) was determined dideoxynucleotides by the method of Sanger (Sanger) using a kit for sequencing AmpliTaq DNA polymerase FS (Cat # 402079 PE Applied Biosystems, Warrington, UK) and apparatus for automatic sequencing Applied Biosystems AB1373A in accordance with the recommendations of the supplier. The initial reaction sequencing was performed using the "direct" and "reverse" -inago primers M13. Subsequent sequencing reactions were carried out in accordance with the method of "DNA-Walking". The oligonucleotides required for subsequent sekvenirovanie, were synthesized by Life Technologies (Inchinnan Business Park, Paisley, UK).

Jghtltktyyst sequences were assembled and analyzed using version 3.2 of the computer program MacDNASIS (Cat. # 22020101, Appligene, Durham, UK). Different open reading frames were analyzed using BLAST algorithm available on the server of the National center for biotechnology information (NCBI, Bethesda, MD, USA).

The full sequence (fragment EcoRI-EcoRI), originally obtained from clone pGEM-7/8 (PLACENTAS. No. 6), presented on Fig.6. She randomly starts after G EcoRI site and shows some uncertainty from the point of view of nucleotides.

Then sequencing optimized, and PLACENTAS. No. 3 (figure 3) gives the General sequence of this strain, which was obtained in such a way that it randomly started from the EcoRI site, i.e. G is used as the first nucleotide.

Similarly, the received sequence of the other three isolates in accordance with the present invention (see PLACENTAS. No. 1, 2 and 4 and figures 1, 2 and 4).

The size of the genome of these four strains is as follows:

1767 nucleotides
Imp.1011-481211767 nucleotides
Imp.1011-48285
Imp.9991768 nucleotides
Imp.10101768 nucleotides

EXAMPLE 11. Sequence analysis of strain PCV Imp.999.

Analysis of the homology sequence obtained from a strain Imp.999, in relation to sequences contained in the GenBank data Bank showed that the only identified significant homology was approximately 76%homology (at the level of the nucleic acid) sequence of strain RK/15 (collectible non Y09921 and U49186) (see figure 5).

At the amino acid level test for homology with the translation of sequences in 6 phases with data banks (the BLAST algorithm X on the NCBI server) demonstrated 94%homology with an open reading frame corresponding to theoretical replicate wins including bbtv virus-like circoviruses plants (identification number of the GenBank data Bank - 1841515)encoded by the sequence U49186 from the database GenBank.

No other sequences available in data banks, did not show significant homology with the sequence obtained from a strain of PCV Imp.999.

Analysis of the sequences obtained from strain Imp.999, the cultivated areas of diseased organs and tissues taken from California pigs with clinical signs multiboot wasting-sin is Roma, clearly shows that this viral isolate represents a new strain of swine circovirus.

EXAMPLE 12. Comparative sequence analysis

Conducted a comparison of the nucleotide sequences of four new strains of PCV with the sequence of strain PCV PK/15 (figure 5). Developed a matrix of homology, which includes four new strain and preceding strain PK/15. Got the following results:

1: Imp.1011-48121

2: Imp.1011-48285

3: Imp.999

4: Imp.1010

5: RK/15

12345
11,00000,99770,96150,96210,7600
21,00000,96210,96320,7594
31,00000,99490,7560
41,00000,7566
51,0000

Homology between the two French strains Imp.1011-48121 and Imp.1011-48285 more than 99% (0,9977).

Homology between the two North American shtam the AMI Imp.999 and Imp.1010 also higher than 99% (0,9949). Homology between the French and North American strains strains slightly above 96%.

The homology between these strains and RK/15 is in the range between 75 and 76%.

From this it follows that the strains of the present invention represent a new type of swine circovirus other than the type represented by strain RK/15. This new type isolated from pigs exhibiting the PMWS syndrome, called swine circovirus type II, and RK/15 represents the type I. the Strains belonging to type II, have shown considerable homogeneity of nucleotide sequences, although in fact they were isolated from the samples received from very remote from each other geographic regions.

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

The nucleotide sequence of the isolate Imp.1010 recognized representative of other strains of circovirus associated with the multiboot wasting syndrome. This sequence was analyzed in more detail using the BLASTX algorithm (Altschul et al., J. Mol. Biol., 1990, 215, 403-410), and using a combination of programs from the software bundle MacVector of 6.0 (Oxford Molecular Group, Oxford OH 4GA, UK). Ha this sequence (circular genome) failed to detect 13 open reading frames (ORF) larger than 20 amino acids. These 13 open reading frames:

NameStartEndChainThe size of the ORF (nucleotide (NT)The size of the proteins (amino acids) (AK)
ORF1103210Semantic10835
ORF211801317Semantic13845
ORF313631524Semantic16253
ORF43981342Semantic945314
ORF59001079Semantic18059
ORF612541334Semantic8126
ORF71018704Antisense315104
ORF8439311Antisense12942
ORF9190101Antisense9029
ORF10912733Antisense18059
ORF11 645565Antisense8126
ORF1211001035Antisense6621
ORF133141381Antisense702213

The position of the beginning and end of each ORF (open reading frame) refer to the sequence represented in figure 4 (PLACENTAS. No. 4), the genome of strain 1010. Border ORF1 to ORF13 identical to strain 999. They are identical to strains 1011-48121 and 1011-48285 except ORF3 and ORF13:

ORF3 1432-1539, semantic, 108 nt, 35 AA

ORF13 314-1377, antisense, 705 nt, 234 AA.

Of these thirteen ORF four have significant homology with the same ORF, located on the genome of the cloned virus PCV RK-15. Analyzed each of the open reading frames present in the genome of all isolates circovirus associated with the multiboot wasting syndrome. These four ORFS following:

NameStartEndChainThe size of the ORF (NT)The size of the protein (AK)Molecular mass (kDa)
ORF43981342semantic945314 37,7
ORF71018704antisense31510411,8
ORF10912733antisense180596,5
ORF133141381antisense70223327,8

The position of the beginning and end of each ORF refer to the sequence represented in figure 4 (PLACENTAS. No. 4). The size of the ORF (nucleotides - NT) includes the termination codon.

Comparison of the organization of the genomes of isolates of PCV Imp.1010 and PCV RK-15 allowed us to identify 4 reading frames, preserved in the genomes of the two viruses. The following table shows the observed degree of homology:

ORF Imp.1010/ORF PVC PK-15The percentage homology
ORF4/ORF186%
ORF13/ORF266,4%
ORF7/ORF361,5% (at the level of overlap (104 AK))
ORF10/ORF483% (at the level of overlap (59 AK))

The greatest sequence identity was observed between ORF4 Imp.1010 and ORF1 RK-15 (homology of 86%). This was to be expected, since this protein apparently involved in replication Viru is Noah DNA 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).

The sequence identity between ORF13 Imp.1010 and ORF2 RK-15 less (homology 66,4%), but each of these two ORFS really shows well-preserved N-terminal basic region, identical to the N-concava main structural protein of circovirus birds CAV (Meehan et al., Arch. Virol., 1992, 124, 301-319). In addition, we observed large differences between ORF7 Imp.1010 and ORF3 RK-15 and between ORF10 Imp.1010 and ORF4 RK-15. In each case, there is a deletion in the C-terminal region ORF7 and ORF10 protein isolate Imp.1010, if to compare them with ORF3 and ORF4 strain PCV RK-15. The highest homology of sequences observed at the level of the N-terminal regions ORF7/ORF3 (homology to 61.5% at the level of overlap) and ORF10/ORF4 (homology of 83% at the level of overlap).

It was found that the genomic organization of circovirus pigs is quite complicated, as a consequence of the extreme density of its genome. The main structural protein, apparently, is the result of splicing between multiple reading frames located on the same circuit circovirus genome of pigs. So we can assume that any open reading frame (ORF1 to ORF13)described in the above table, may constitute the entire antigenic protein or portion of this protein, encoded by swine circovirus type II, and therefore is potentially a antigen, which is to use to establish a specific diagnosis and/or for vaccination. Therefore, the invention relates to any protein that includes at least one of these open reading frames. Preferably, the invention relates to protein, essentially consisting of ORF4, ORF7, ORF10 or ORF13.

EXAMPLE 14. The infectious nature of the PCV genome, cloned from new strains.

The plasmid pGEM-7/8 containing the full genome (replicative form) isolate Imp.999, transferrable cells RK/15 in accordance with the method described Meehan C. 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 assay (see example 4), performed at the first passage after transferowania on decontaminating cells RK/15 showed that a plasmid clone pGEM7/8 is able to induce the production of infectious virus PCV. The presence of a clone containing infectious genetic material PCV, allows you to perform any useful manipulation of the viral genome in order to produce modified viruses PCV (or attenuated in pigs or defective), which can be used to generate attenuated or recombinant vaccines or for the production of antigens for diagnostic kits.

EXAMPLE 15. Production of antigens PCV by in vitro cultivation.

Cultivation decontaminating cells RK/15 and reproduction in the Russes was carried out in the same manner described in example 1. Infecting the cells were collected after trypsinization, after 4 days incubation at 37°and counted. The following passage was inoculable inficirovannymi cells with a concentration of 400,000 living cells in 1 ml

EXAMPLE 16 Inactivation of viral antigens

At the end of cultivation of viruses infecting cells were collected and literally using ultrasound (Branson Sonifer) or by using a colloid mill rotor-stator type (UltraTurrax, IKA). Then the suspension was centrifuged at 3700 g for 30 minutes. Suspension of viruses iactiveaware 0,1%ethylenimine for 18 hours at +37°or 0.5%beta-propiolactone within 24 hours at +28°C. If the titer of the virus before inactivation was insufficient, the suspension of the virus was concentrated by ultracentrifugation using a membrane permeable connection up to 300 kDa (Millipore RTMC). Inactivated suspension of virus was stored at +5°C.

EXAMPLE 17 Receiving the vaccine in the form of an emulsion based on mineral oil

The vaccine was received in accordance with the following formula:

Suspension of inactivated swine circovirus250 ml
Montanide®ISA 70 (SEPPIC)750 ml

The aqueous phase and the oil phase sterilized is whether separately by filtering. The emulsion was prepared by mixing and homogenization of the ingredients with the help of turbine emulsifier Silverson model.

One dose of the vaccine contains approximately 107,5TCID50. The volume of one dose of vaccine is 0.5 ml for intradermal and 2 ml for intramuscular injection.

EXAMPLE 18 Receiving the vaccine in the form of metabolisable emulsion oil-based

The vaccine was received in accordance with the following formula:

Suspension of inactivated swine circovirus200 ml
Dehymuls HRE 7 (Henkel)60 ml
Radia 7204 (Oleofina)740 ml

The aqueous phase and oil phase were sterilized separately by filtration. The emulsion was prepared by mixing and homogenization of the ingredients with the help of turbine emulsifier Silverson model.

One dose of the vaccine contains approximately 107,5TCID50. The volume of one dose of vaccine is 2 ml for intramuscular injection.

EXAMPLE 19 the Results of indirect fluorescent analysis of virus strains PCV from the USA and France and RK/15 with hyperimmune serum (PCV-T), with a set of monoclonal antibody F99 obtained from RK/15, and hyperimmune serum obtained from the canadian strain (PCV-C)

VI THE US
RK/15USAFrance
Anticavity PCV-T≥ 6400200800
Anticavity PCV-C200≥ 6,400≥ 6,400
F99 N≥ 10000< 100100
F99 V≥ 10000< 100< 100
F99 V≥ 10000100< 100
F99 E≥ 10000< 100< 100
F99 S≥ 10000< 100100
F99 2E1≥ 10000< 100< 100
F99 N≥ 10000100< 100
* When interacting with the last dilution of serum and a monoclonal antibody, giving a positive reaction in the indirect immunofluorescence assay.

1. The selected strain of swine circovirus type II, which is the causative agent paleochannel Multisystem wasting syndrome (PMWS), deposited in ESAS under deponent number V97100219.

2. The selected strain of swine circovirus type II, which is the causative agent polythene what about the Multisystem wasting syndrome (PMWS), deposited in ESAS under deponent number V97100218.

3. The selected strain of swine circovirus type II, which is the causative agent paleochannel Multisystem wasting syndrome (PMWS), deposited in ESAS under deponent number V97100217.

4. The selected strain of swine circovirus type II, which is the causative agent paleochannel Multisystem wasting syndrome (PMWS), deposited in ESAS under deponent number V98011608.

5. The selected strain of swine circovirus type II, which is the causative agent paleochannel Multisystem wasting syndrome (PMWS), deposited in ESAS under deponent number V98011609.

6. The selected DNA fragment comprising a sequence selected from the group consisting of SEQ ID No 1, 2, 3, 4, and 6, representing a genomic sequence of the swine circovirus type II according to claims 1 to 5, respectively.

7. The selected DNA fragment according to claim 6, comprising SEQ ID No 1, representing a genomic nucleotide sequence of a selected strain of swine circovirus type II V98011609 according to claim 5.

8. The selected DNA fragment according to claim 6, comprising SEQ ID No 2, representing a genomic nucleotide sequence of a selected strain of swine circovirus type II V98011608 according to claim 4.

9. The selected DNA fragment according to claim 6, comprising SEQ ID No 3, representing a genomic nucleotide sequence of the separation of the spent strain of swine circovirus type II V97100217 according to claim 3.

10. The selected DNA fragment according to claim 6, comprising SEQ ID No 4, representing a genomic nucleotide sequence of a selected strain of swine circovirus type II V97100218 according to claim 2.

11. The selected DNA fragment according to claim 6, comprising SEQ ID No 6, representing a genomic nucleotide sequence of a selected strain of swine circovirus type II V97100219 according to claim 1.

12. The selected DNA fragment comprising the nucleotide sequence encoding the epitope specific for PCVII and not specific to PCVI.

13. Viral preparation containing swine circovirus type II caller paleochannel multi-systemic wasting syndrome (PMWS), a nucleotide sequence which's hybrid with genomic nucleotide sequence of SEQ ID No 1, 2, 3, 4, or 6 on article 6 of swine circovirus type II according to any one of claims 1 to 5, or a fragment, in the strict conditions under which no hybridization with the genomic nucleotide sequence PCVI SEQ ID No 5.

14. The viral preparation according to item 13, characterized in that it is grown in cell culture in vitro, which were infected by a strain of swine circovirus type II isolated from samples taken from pigs with PMWS.

15. The viral preparation according to 14, characterized in that the sample comes from the affected tissue.

16. The viral preparation according to 14 or 15, characterized in that the cells are is a line of renal cells of the pig.

17. The viral preparation according to item 16, characterized in that the line of the renal cells of the pig represents RK/15.

18. The viral preparation according to any one of p-17, which is weakened.

19. The viral preparation according to any one of p-17, which is inaktivirovannye.

20. Viral preparation containing swine circovirus type II caller paleochannel multi-systemic wasting syndrome (PMWS), genomic nucleotide sequence which is at 96% or more homologous to the genomic nucleotide sequence of SEQ ID No 1, 2, 3, 4, or 6 on article 6 of swine circovirus type II according to any one of claims 1 to 5.

21. The viral preparation according to claim 20, characterized in that it is grown in cell culture in vitro, which were infected by a strain of swine circovirus type II isolated from samples taken from pigs with PMWS.

22. The viral preparation according to item 21, characterized in that the sample comes from the affected tissue.

23. The viral preparation according to item 21 or 22, characterized in that the cells are a line of renal cells of the pig.

24. The viral preparation according to item 23, characterized in that the line of the renal cells of the pig represents RK/15.

25. The viral preparation according to any one of p-24, which is weakened.

26. The viral preparation according to any one of p-24, which is inaktivirovannye.

27. The viral preparation, stereoselectivity pigs type II, genomic nucleotide sequence which is at 76% or less homologous to the genomic nucleotide sequence of the PCVI SEQ ID No 5.

28. The viral preparation according to item 27, characterized in that it is grown in cell culture in vitro, which were infected by a strain of swine circovirus type II isolated from samples taken from pigs with PMWS.

29. The viral preparation according p, characterized in that the sample comes from the affected tissue.

30. The viral preparation according p or 29, characterized in that the cells are a line of renal cells of the pig.

31. The viral preparation according to item 30, characterized in that the line of the renal cells of the pig represents RK/15.

32. The viral preparation according to any one of p-31, which is weakened.

33. The viral preparation according to any one of p-31, which is inaktivirovannye.

34. Viral preparation containing swine circovirus type II, which is the causative agent paleochannel Multisystem wasting syndrome (PMWS), a nucleotide sequence which is at 96% or more homologous to the genomic nucleotide sequence of SEQ ID No 1, 2, 3, 4, or 6 on article 6 of swine circovirus type II according to any one of claims 1 to 5, and 76% or less homologous to the genomic nucleotide sequence of the PCVI SEQ ID No 5.

35. The viral preparation according to 34, characterized in that it is grown in cultures of the cells in vitro, who have been infected with a strain of swine circovirus type II isolated from samples taken from pigs with PMWS.

36. The viral preparation according p, characterized in that the sample comes from the affected tissue.

37. The viral preparation according p or 36, characterized in that the cells are a line of renal cells of the pig.

38. The viral preparation according to clause 37, characterized in that the line of the renal cells of the pig represents RK/15.

39. The viral preparation according to any one of p-38, which is weakened.

40. The viral preparation according to any one of p-38, which is inaktivirovannye.

41. Viral preparation containing swine circovirus type II genomic nucleotide sequence which encodes proteins ORF 4, 13, 7, and 10, with protein ORF 4 86% or less homologous protein ORF 1 PCVI, protein ORF 13 by 66.4% or less homologous protein ORF 2 PCVI, protein ORF 7 61,5% or less homologous protein ORF 3 PCVI and protein ORF 10 83% or less homologous protein ORF 4 PCVI.

42. The viral preparation according to paragraph 41, characterized in that it is grown in cell culture in vitro, which were infected by a strain of swine circovirus type II isolated from samples taken from pigs with PMWS.

43. The viral preparation according to § 42, characterized in that the sample comes from the affected tissue.

44. The viral preparation according to § 42 or 43, characterized in that the cell represents the line of the renal cells of the pig.

45. The viral preparation according to item 44, characterized in that the line of the renal cells of the pig represents RK/15.

46. The viral preparation according to any one of p-45, which is weakened.

47. The viral preparation according to any one of p-45, which is inaktivirovannye.

48. Cell extract, calling paleochannel multi-systemic wasting syndrome (PMWS), characterized in that the extract was obtained from the culture of cells in vitro, which was infected with swine circovirus type II according to any one of claims 1 to 5 or a viral preparation according to any one of p-47.

49. The supernatant of cell culture, calling paleochannel multi-systemic wasting syndrome (PMWS), characterized in that the above-mentioned supernatant was obtained from the culture of cells in vitro, which was infected with swine circovirus type II according to any one of claims 1 to 5 or a viral preparation according to any one of p-47.

50. Immunogenic composition comprising a selected strain of swine circovirus type II according to any one of claims 1 to 5, the viral preparation according to any one of p-47, cell extract by p or supernatant of the cell culture according to § 49 and a pharmaceutically acceptable carrier.

51. Immunogenic composition according to item 50, containing the swine circovirus type II.

52. Immunogenic composition according to item 50, containing an inactivated swine circovirus type II.

53. Immunogenic component is ice on any of PP-52, which includes strain of swine circovirus type II, propagated in cell lines.

54. Immunogenic composition according to item 53, in which the cell line is a line of renal cells of the pig.

55. Immunogenic composition according to § 51, comprising from 103up to 106TCID50attenuated swine circovirus type II.

56. Immunogenic composition according to § 55, comprising from 106up to 108TCID50strain of swine circovirus type II.

57. Immunogenic composition according to any one of p-56, manufactured in liofilizovannyh form.

58. Immunogenic composition according to § 57, further comprising a stabilizer lyophilization.

59. Immunogenic composition according to § 58, in which the stabilizer lyophilization selected from the group consisting of SPGA, sorbitol, mannitol, starch, sucrose, dextran, glucose, albumin, casein and phosphate of an alkali metal.

60. Immunogenic composition according to any one of p-59, further comprising adjuvant.

61. Immunogenic composition according p, in which the adjuvant is selected from the group consisting of aluminium hydroxide, saponin, avridine (N,N-dioctadecyl-N',N'-bis(2-hydroxyethyl)propandiamine), DDA.

62. Immunogenic composition according to any one of p-59, made in the form of an emulsion.

63. Immunogenic composition according to item 62, in which the emulsion is an emulsion of water-in-oil.

64. Immunogenic to notice according to item 62, in which the emulsion is an emulsion of oil-in-water.

65. Immunogenic composition according to any one of p-64, including a concentrated culture of swine circovirus type II.

66. Immunogenic composition according to paragraph 52, in which the swine circovirus was inactivated by chemical means.

67. Immunogenic composition according p, in which the chemical agent is selected from the group consisting of formaldehyde, paraformaldehyde, β-propiolactone and ethylenimine.

68. Immunogenic composition according to any one of p-67, further comprising another pathogen of pigs.

69. Immunogenic composition according p, in which an additional pathogen of pigs selected from the group consisting of a virus reproductive and respiratory syndrome swine (PRRS), Mycoplasma hyopneumoniae, Actinobacillus pleuropneumoniae, Escherichia coli, Pasteurella multocida (causes atrophic rhinitis), virus false rabies (causes Aujeszky disease)virus, swine fever (causes hog cholera and swine influenza virus.

70. Immunogenic composition according p, in which an additional pathogen of pigs is a PRRS virus.

71. Immunogenic composition according p, in which an additional pathogen of pigs is a Mycoplasma hyopneumoniae.

72. Immunogenic composition according to any one of p-71, which is a vaccine.

73. Immunogenic composition, including any subunit is dedicated to the swine circovirus type II.

74. Immunogenic composition according p, in which the subunit obtained by expression in vitro.

75. Immunogenic composition according p, in which the subunit expressed using baculovirus.

76. Immunogenic composition according p, in which the subunit expressed in E. coli.

77. Immunogenic composition according p, in which the subunit obtained by expression in eukaryotic cells.

78. Immunogenic composition according p, in which the eukaryotic cells are yeast cells.

79. Immunogenic composition according p, in which yeast cells are S. cerevisiae.

80. Immunogenic composition according p, in which the eukaryotic cells are mammalian cells.

81. Immunogenic composition according to item 80, in which the mammalian cells are selected from the group consisting of cells, Cho cells and KSS.

82. Immunogenic composition according to any one of p-81, in which the subunit in vitro by expression of any open reading frame of swine circovirus type II.

83. Immunogenic composition according p, in which the open reading frame is a ORF4.

84. Immunogenic composition according p, in which the open reading frame is a ORF7.

85. Immunogenic composition according p, in which the open reading frame is a ORF10.

86. Immune the genetic composition p, in which the open reading frame is a ORF13.

87. Immunogenic composition according to any one of p-86, further comprising adjuvant.

88. Immunogenic composition according to any one of p-86, further comprising another pathogen of pigs.

89. Immunogenic composition according p, in which an additional pathogen of pigs selected from the group consisting of a virus reproductive and respiratory syndrome swine (PRRS), Mycoplasma hyopneumoniae, Actinobacillus pleuropneumoniae, Escherichia coli, Pasteurella multocida (causes atrophic rhinitis), virus false rabies (causes Aujeszky disease)virus, swine fever (causes hog cholera and swine influenza virus.

90. Immunogenic composition according p, in which an additional pathogen of pigs is a PRRS virus.

91. Immunogenic composition according p, in which an additional pathogen of pigs is a Mycoplasma hyopneumoniae.

92. Immunogenic composition according to any one of p-91 comprising an inactivated or attenuated PCVII or subunit PCVII from more than one strain of swine circovirus type II.

93. Immunogenic composition according to any one of PP-92, which is a vaccine.

94. Immunogenic composition comprising a vector that contains and expresses in vivo in the cells of the pig-the owner of the fragment of the nucleic acid or its fragment that encodes a polypeptide or peptide pig circovirus the th type II, and pharmaceutically acceptable carrier or diluent.

95. Immunogenic composition according p, in which the vector is a viral vector.

96. Monogenea composition on p, in which the vector is a plasmid.

97. Immunogenic composition according p, which induces an immunological response in the pig host against swine circovirus type II.

98. Immunogenic composition according p-97, further comprising one another antigen of a pathogen of pigs.

99. Immunogenic composition according p, in which the additional antigen is from a pathogen of pigs selected from the group consisting of a virus reproductive and respiratory syndrome swine (PRRS), Mycoplasma hyopneumoniae, Actinobacillus pleuropneumoniae, E. coli, atrophic rhinitis of pigs, false rabies (Aujeszky disease), hog cholera and swine flu.

100. Immunogenic composition according p, in which the additional antigen is from PRRS.

101. Immunogenic composition according p, in which the additional antigen is from Mycoplasma hyopneumoniae.

102. Immunogenic composition according to any one of PP-101, in which the vector expresses a polypeptide or peptide comprising at least one immunogen or one epitope strain of swine circovirus type II.

103. Immunogenic composition according to any one of p-102, in which the vector expresses the polypeptides or peptides of the more che is one strain of swine circovirus type II.

104. Immunogenic composition according to any one of p-103, which is a vaccine.

105. A vector comprising a selected DNA fragment containing

(a) the sequence shown in SEQ ID No 1, 2, 3, 4, or 6, or

(b) fragment that encodes a polypeptide or peptide containing virousspecificakih epitope, and the specified portion (b) 's hybrid sequence (a) under strict conditions under which no hybridization with the genomic nucleotide sequence PCVI.

106. Vector on p, in which the DNA fragment comprises SEQ ID No 1.

107. Vector on p, in which the DNA fragment comprises SEQ ID No 2.

108. Vector on p, in which the DNA fragment comprises SEQ ID No 3.

109. Vector on p, in which the DNA fragment comprises SEQ ID No 4.

110. Vector on p, in which the DNA fragment comprises SEQ ID No 6.

111. The vector according to any one of p-110, which expresses the polypeptide or peptide comprising at least one immunogen or one epitope strain of swine circovirus type II.

112. The vector according to § 111, which provides the expression embedded in his DNA fragment in vitro.

113. The vector according to § 111, which provides the expression embedded in his DNA fragment in vivo.

114. Vector on p, which is a plasmid.

115. Vector on p representing the virus.

116. Vector on p, in which the virus is selected from the group sotoyama the pork herpesvirus, porcine adenovirus and poxvirus.

117. Vector on p in which the virus is selected from the group consisting of virus false rabies (Aujeszky disease), vaccinia virus, pox virus of birds and pox virus swine.

118. Vector on p in which the virus is a pox virus Canaries.

119. A vector comprising a selected DNA fragment containing a sequence selected from the group consisting of ORF 4, 7, 10 and 13 of swine circovirus type II.

120. Vector on p, whose ORF represents the ORF 4.

121. Vector on p, whose ORF represents the ORF 7.

122. Vector on p, whose ORF represents the ORF 10.

123. Vector on p, whose ORF represents the ORF 13.

124. The vector according to any one of p-123, which provides the expression embedded in his DNA fragment in vitro.

125. The vector according to any one of p-123, which provides the expression embedded in his DNA fragment in vivo.

126. Vector on p, which is a plasmid.

127. Vector on p representing the virus.

128. Vector on p in which the virus is selected from the group consisting of pork herpesvirus, porcine adenovirus and poxvirus.

129. Vector on p in which the virus is selected from the group consisting of virus false rabies (Aujeszky disease), vaccinia virus, pox virus of birds and pox virus swine.

130. Vector on p, is where the virus is a pox virus Canaries.

131. A vector comprising a selected DNA fragment according to item 12.

132. Vector on p providing expression embedded in his DNA fragment in vitro.

133. Vector on p providing expression embedded in his DNA fragment in vivo.

134. Vector on p, which is a plasmid.

135. Vector on p representing the virus.

136. Vector on p in which the virus is selected from the group consisting of pork herpesvirus, porcine adenovirus and poxvirus.

137. Vector on p in which the virus is selected from the group consisting of virus false rabies (Aujeszky disease), vaccinia virus, pox virus of birds and pox virus swine.

138. Vector on p in which the virus is a pox virus Canaries.

139. Immunogenic composition comprising a vector according to any one of p-118.

140. Immunogenic composition according p, which is a vaccine.

141. Immunogenic composition comprising a vector according to any one of p-130.

142. Immunogenic composition according p, which is a vaccine.

143. Immunogenic composition comprising a vector according to any one of p-138.

144. Immunogenic composition according p, which is a vaccine.

145. Selected virousspecificakih polypeptide of swine circovirus type II encoded by the DNA fragment according to any one of p-12 or expressed by a vector according to any one of p-138.

146 Immunogenic composition, including the selected polypeptide according p.

147. Immunogenic composition according p, which is a vaccine.

148. Polyclonal or monoclonal antibody, which is induced by or associated with a strain of swine circovirus type II according to any one of claims 1 to 5, a viral preparation according to any one of p-47, cell extracts by p, the supernatant according to § 49 or dedicated polypeptide ω p.

149. Method of inducing immunological response or induction of antibodies against swine circovirus type II, including the introduction of the pig immunogenic composition according to any one of p-104, 139-144 and 146-147.

150. The method for detecting swine circovirus type II in a sample, comprising testing the sample for the presence of the antigen of swine circovirus type II, or for the presence of antibodies against swine circovirus type II or against the antigen of swine circovirus type II, or nucleic acid of swine circovirus type II.

151. The method of obtaining strain of swine circovirus type II according to claims 1 to 5, the viral preparation according to any one of p-47, cellular drug p or supernatant in § 49, which includes the cultivation and selection of cell culture in vitro of cells that have been infected with a strain of swine circovirus type II.

152. Cell extract containing the strain of swine circovirus type II, obtained in accordance with the method according to p that mo is should be used as antigen.

153. The cell supernatant containing the strain of swine circovirus type II, obtained in accordance with the method according to p, which can be used as antigen.

154. The antibody is able to connect with swine circovirus type II, obtained by the method according to p.

155. A method for the diagnosis of infection with swine circovirus type II (PCVII)causing paleochannel multi-systemic wasting syndrome (PMWS), which includes the contacting of the sample of physiological fluid or tissue of pigs with a diagnostic reagent that is specific to a PCVII, and the detection of the presence or absence in the sample or tissue antigen PCVII or antibodies specific to a PCVII, or nucleic acids that are specific to a PCVII.

156. The method according to p, in which the diagnostic reagent comprises at least one DNA probe or primer specific to PCVII.

157. The method according to p, in which the diagnostic reagent comprises SEQ ID No 1, 2, 3, 4, or 6.

158. The method according to p, in which the probe or primer comprises at least one fragment of sequence SEQ ID No 1, 2, 3, 4, or 6.

159. The method according to any of PP-158, in which detection is the detection of nucleic acid molecules that are specific to a PCVII, hybridization or PCR.

160. The method according to p, in which the diagnostic reagent comprises an antigen specific for PCVII.

161. The method according to p, in which the antigen, specific for PCVII includes a selected strain of swine circovirus type II according to any one of claims 1 to 5, the viral preparation according to any one of p-47, cell extract by p or supernatant of the cell culture according to § 49.

162. The method according to p, in which the diagnostic reagent comprises an epitope or polypeptide specific for PCVII and encoded by the fragment of sequence SEQ ID No 1, 2, 3, 4, or 6.

163. The method according to p or 162, in which detection is the detection of antibodies specific to a PCVII.

164. The method according to p, in which the diagnostic reagent comprises an antibody specific for PCVII.

165. The method according to p, in which the diagnostic reagent comprises an antigen specific for PCVII, and the antibody specific to PCVII.

166. The method according to any of PP-165, in which the detecting includes western blot, immunofluorescence, ELISA or immunochromatography.

167. The method according to p, in which the detecting includes indirect immunofluorescence, competitive option ELISA or immunochromatography.

168. The diagnostic composition comprising an antibody specific to the strain of swine circovirus type II (PCVII)responsible for paleochannel multi-systemic wasting syndrome (PMWS), which is obtained from PCVII or antigenic fragment, or polypeptide fragment encoded by the sequence SEQ ID No 1, 2, 3, 4, or 6.

169. The composition according to p to the ora PCVII is a deposited selected strain of swine circovirus type II according to any one of claims 1 to 5 or the viral preparation according to any one of p-47.

170. The composition according to p or 169, in which the antibodies are polyclonal antibodies or monoclonal antibodies.

171. Composition according to any one of p-170, in which antibodies are labeled antibody.

172. The composition according to p, in which antibodies in the state of peroxidase or a label in the form of particles.

173. The composition according to p, in which the label is in the form of particles represents the label of the colloidal gold.

174. A diagnostic composition comprising the antigen-specific strain of swine circovirus type II (PCVII)responsible for paleochannel multi-systemic wasting syndrome (PMWS), which is recognized by antibodies specific to a PCVII.

175. Diagnostic kit for the diagnosis of swine circovirus type II caller PMWS, comprising at least one composition according to any one of p-174.

176. Set p, optionally including western blot, immunofluorescence, ELISA or immunochromatography as diagnostic tools.

177. Set p, including means for indirect immunofluorescence assay, a competitive variant of ELISA or chromatography.

178. Virousspecificakih means causing paleochannel multi-systemic wasting syndrome (PMWS)containing the selected strain of swine circovirus type II according to any one of claims 1 to 5, the viral preparation according to any one of p-47, cell extract by p or 148 or su is inatant cell culture according to § 49 or 153.

Priority areas;

03.10.1997 - claims 1 to 3, 9, 12, 27, 131-138, 143-144, 150, 155-156, 160, 164-167;

22.01.1998 - claims 4-8, 10-11, 13, 20, 34, 105-118, 139-142, 157-159, 168;

20.03.1998 - PP-19, 21-26, 28-33, 35-68, 73-88, 94-98, 119-130, 145-149, 151-154, 161-163, 169-178;

01.10.1998 - PP-72, 89-93, 99-104.



 

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