RU2803427C1 - Immunogenic compositions for immunization of pigs against type 3 circovirus and methods of their production and use - Google Patents

Abstract

FIELD: biotechnology and veterinary medicine.

SUBSTANCE: method of inducing an immunological response against type 3 porcine circovirus is proposed, the method includes the following steps: administering to an animal of a composition containing a protein with an amino acid sequence having at least 90% sequence identity with SEQ ID NO. 6, or containing a protein encoded by a nucleotide sequence having at least 90% sequence identity with SEQ ID NO. 5, or a composition containing a vector with a heterologous nucleotide sequence encoding a protein with an amino acid sequence having at least 90% sequence identity with SEQ ID NO. 6, or containing a vector with a heterologous nucleotide sequence having at least 90% sequence identity with SEQ ID NO. 5, or a vector with a heterologous RNA sequence corresponding to a nucleotide sequence having at least 90% sequence identity with SEQ ID NO. 5.

EFFECT: invention makes it possible to induce an immunological response against type 3 porcine circovirus.

53 cl, 2 dwg, 6 ex

Description

[1] This application claims the benefit of U.S. Provisional Patent Application No. 62/248,666, filed Oct. 16, 2015, which is incorporated herein by reference in its entirety.

LIST OF SEQUENCES

[2] The application contains a sequence listing presented in paper and machine-readable format, the data and contents of which are incorporated herein by reference.

BACKGROUND OF THE ART

[3] Porcine circovirus type 2 (PCV2) was first sporadically identified in pigs with post-weaning multisystem wasting syndrome (PMWS) in Canada in the mid-1990s. Epidemics of severe systemic disease associated with PCV2 were subsequently identified in Europe and Asia, and subsequently in North America. Widespread use of commercial vaccines has effectively controlled PCV2-associated disease (PCVAD), which includes PMWS, pneumonia, porcine dermatitis and nephropathy syndrome (PDNS), and reproductive failure. Retrospective studies have demonstrated that PCV2 circulated undetected in pigs for several decades before becoming a widespread clinical disease. A change in the dominant genotype from PCV2a to PCV2b appears to correspond to severe PCVAD. Recently, a new species of circovirus, named porcine circovirus type 3 (PCV3), was identified in sows with clinical symptoms typically associated with PCV2 infection; this type of circovirus has also been identified in aborted fetuses.

SUMMARY OF THE INVENTION

[4] Tissues were collected from four sows from a farm with chronic low fertility that died suddenly and were accompanied by clinical symptoms consistent with PDNS. Pathological examination of porcine tissues revealed violaceous skin lesions with acute necrotic lesions of the dermis and epidermis associated with hemorrhage and perivascular collections of leukocytes. In the kidneys, dilated renal tubules with clusters of lymphocytes and macrophages in the interstitium and glomeruli and mild dysplasia in tubular epithelial cells were observed. Despite the macroscopic and microscopic lesions typically seen with PCV2 infection, immunohistochemistry and quantitative PCR (qPCR) were negative for the presence of PCV2 as well as porcine reproductive and respiratory syndrome virus (PRRSV) and influenza A virus (IAV). At the same time, mummified abortions from sows with similar skin lesions were collected from the same farm. qPCR results were also negative for PCV2, as well as porcine reproductive and respiratory syndrome virus (PRRSV) and porcine parvovirus (PPV).

[5] Pooled tissue homogenates were obtained from three mummies, and sequencing of the viral metagenomes described above was performed. Of the 989,478 reads collected, 926,380 were mapped to the Sus scrofa reference genome. De novo assembly of ungrouped reads resulted in 27 contigs (sets of overlapping sequences). The majority (54%) of the reads mapped to a 1,246-bp contig, which by BLASTN analysis had 98% homology to the partial circovirus genome found in commercial porcine meat subjected to metagenomic sequencing. The remaining reads showed no similarity to known eukaryotic viruses. This was followed by spin-circle amplification of the DNA pool obtained from mummies, followed by reverse PCR using primers spanning the XhoI site contained in the circovirus contig. The results of agarose gel electrophoresis showed the presence of a single band of approximately 2 kb. Genome 2000 bp long. (SEQ ID No.1) was determined by Sanger sequencing using overlapping amplicons covering the entire genome.

[6] Genetic analysis revealed an open reading frame (ORF1, SEQ ID No.3) encoding a predicted protein of 296 amino acids (aa) in length (SEQ ID No.4), which, according to BLASTP analysis, was 96% identical to the partial protein, replicase (rep), circovirus PorkNW2/USA/2009 (accession number ADU77001, 221 aa) and 54% identical to the 293 aa rep protein identified in China from bat circovirus (accession number AIF76248). The conserved viral rep and helicase domains were identified as aa 9-93 and 162-251 of SEQ ID No. 1, respectively. Similar to PorkNW2/USA/2009, a suitable start codon was not identified and an alternative start codon GTC or splicing from the upstream ATG was proposed. The second ORF (ORF2, SEQ ID No.5) in the opposite orientation encoded a predicted 214 aa protein (SEQ ID No.6) 87% identical to the partial sequence (110 aa) of the capsid protein PorkNW2/USA/2009 and 36- 37% identical to the capsid proteins of PCV2 and duck ciroviruses (233 and 257 aa, respectively). Similar to other circovirus cap proteins, the N-terminus consisted of an arginine-rich region approximately 32 aa in length. The third ORF (ORF3, SEQ ID No. 7), encoding a predicted 233 aa protein (SEQ ID No. 8), was 94% identical to the ORF identified in the partial circuvirus genome obtained from ground beef, and 39% identical to the herpes protein Murid with unknown function. The predicted origin of replication contained a stem-loop structure with a nonamer loop identical to that of PCV1, TAGTATTAC (SEQ ID No. 15), located on the coding strand between rep and cap (Fig. 1). Given the overall genetic and structural similarity to members of the genus Circovirus and <70% aa identity of the capsid protein with other species, this species is referred to herein as porcine circovirus type 3 (PCV3).

[7] Phylogenetic analysis was performed in MEGA with sequences aligned using the ClustalW program. Phylogenetic analysis was performed using maximum likelihood best LG fit with a gamma distribution model with tree topology for 500 bootstrap replicates of rep protein sequences (Fig. 2). A scale representing the number of amino acid changes is shown. GenBank accession numbers are given in parentheses. In fig. 2 CaCV is a canary circuvirus; GuCV - gull circuvirus; FiCV - finch circovirus; StCV - starling circuvirus; PiCV - pigeon circovirus; BFDV - beak and feather disease virus; DuCV - duck circovirus; GoCV - goose circovirus; SwCV - swan circovirus; BtCV - bat circovirus; PCV1 - porcine circovirus type 1; and PCV2, porcine circovirus type 2. As shown in the figures, PCV3 clusters with PorkNW2/USA/2009, which is most closely related to bat circovirus from China. Interestingly, PCV1 and PCV2 are also evolutionarily related to another bat circovirus from Myanmar.

[8] Tissue homogenates from three different mummies and lung homogenates from four sows that died of PDNS were analyzed by qPCR targeting the rep gene PCV3. All mummies were positive with cycle threshold (Ct) values of 16.7–21.3. Three out of four sows were also positive with Ct values of 27.7-29.7.

[9] To determine the incidence of PCV3, a total of 271 samples of nasal swabs, oral fluids, or lung homogenates obtained from pigs for diagnostic testing were screened using a multiplexed Taqman assay targeting the rep and cap genes. Thirty-four (12.5%) samples were positive in both assays with similar Ct values. Two samples were positive only by assays targeting cap PCV3. Ct values for positive samples were 20.3–35.8.

[10] The peptide spanning 35-214 aa cap SEQ ID No. 1 was expressed as an N-terminal 6x-histidine recombinant protein in E. coli and purified by affinity chromatography. Denaction electrophoresis showed that the protein was expressed as a dimer, similar to that observed for PCV2. ELISA was performed as described above. Eighteen serum samples from 3-week-old pigs from a defined pathogen-free herd were used as negative controls, with an average optical density of 0.49. The threshold for a positive result was set at three standard deviations above the mean (OD > 0.87). Sera from ten multiparous sows from the same farm were positive with a mean OD value of 1.27. Twenty-seven sera obtained from young farm pigs that were being bred to replace sows were also tested; seventeen (63%) tested positive. Finally, forty-six (55%) of the 83 serum samples (unknown animals) submitted for diagnostic testing from multiple states were positive.

BRIEF DESCRIPTION OF DRAWINGS

[11] The following drawings are part of the present specification and are included to further demonstrate certain aspects of the present invention. The invention may be better understood with reference to one or more of these drawings in combination with the detailed description of the specific embodiments presented in the present description.

Figure 1 is a diagram of the genome organization of porcine circovirus type 3; And

Figure 2 is a phylogenetic tree of circovirus replicase proteins.

[12] In the present application, SEQ ID No. 1 is the nucleotide sequence of the PCV3 genome; SEQ ID No.2 is the amino acid sequence of the PCV3 genome; SEQ ID No. 3 is the nucleotide sequence of PCV3 replicase (ORF1); SEQ ID No.4 is the amino acid sequence of PCV3 replicase (ORF1); SEQ ID No.5 is the nucleotide sequence of PCV3 capsid protein (ORF2); SEQ ID No. 6 is the amino acid sequence of PCV3 capsid protein (ORF2); SEQ ID No. 7 is the nucleotide sequence of PCV3 ORF3; SEQ ID No. 8 is the amino acid sequence of PCV3 ORF3; SEQ ID No.9 is the nucleotide sequence of PCV3 replicase with primer 1; SEQ ID No. 10 is the nucleotide sequence of PCV3 replicase with primer 2; SEQ ID No. 11 is the nucleotide sequence of PCV3 replicase with probe; SEQ ID No. 12 is the nucleotide sequence of the PCV3 capsid protein with primer 1; SEQ ID No. 13 is the nucleotide sequence of the PCV3 capsid protein with primer 2; SEQ ID No. 14 is the nucleotide sequence of the PCV3 capsid protein with probe; SEQ ID No. 15 is an origin of replication containing a stem-loop structure with a nonamer loop; SEQ ID No. 16 is the internal primer of the cap gene; SEQ ID No. 17 is the internal primer of the cap gene; SEQ ID No. 18 is a primer for the portion of the PCV3 gene encoding amino acids (aa) 35-214; and SEQ ID No. 19 is a primer for the portion of the PCV3 gene encoding amino acids (aa) 35-214.

DETAILED DESCRIPTION OF THE INVENTION

[13] The following detailed description and examples set forth preferred materials and procedures used in accordance with the present invention. However, it should be understood that this description and examples are provided by way of illustration only, and should not be construed as limiting the general scope of the present invention.

[14] Immunogenic compositions and methods for preparing and using such compositions

[15] In one aspect, the present invention provides a method for producing and/or recovering recombinant PCV3 ORF2 protein by: 1) infecting multiple susceptible cells in culture with a recombinant viral vector encoding the PCV3 protein, 2) expressing the PCV3 protein using the recombinant viral vector, 3) recovery of PCV3 protein; and 4) separation of cellular debris from expressed PCV3 protein in a separation step.

[16] In another aspect of the present invention, it is preferred to include an inactivation step to inactivate the viral vector prior to recovery of the PCV3 protein to be used in an immunogenic or immunological composition such as a vaccine. Such a step can be performed as step 5) in addition to steps 1-4 described above.

[17] In some forms, this inactivation is carried out either immediately before or immediately after the filtration or separation step. For the purposes of the present invention, any conventional inactivation method can be used. Thus, inactivation can be accomplished by chemical and/or physical methods. One inactivation option involves the addition of cyclized binary ethylenimine (BEI).

[18] The method described above may also optionally include a neutralization step after step 5). For example, if the inactivating agent is BEI, it is preferable to add sodium thiosulfate to an equivalent amount. For inactivation, it is preferable to add an equivalent amount of sodium thiosulfate compared to adding BEI.

[19] “Immunogenic or immunological composition” refers essentially to a composition that contains at least one antigen that induces an immunological response in the host of a cellular and/or antibody-mediated immune response to the composition or vaccine of interest. Typically, an "immunological response" includes, without limitation, one or more of the following effects: production or activation of antibodies, B cells, helper T cells, suppressor T cells and/or cytotoxic T cells and/or yd T cells, directed specifically at the antigen or antigens included in the composition or vaccine of interest. Preferably, the host will exhibit either a therapeutic or a protective immunological response such that resistance to the new infection is increased and/or the clinical severity of the disease is reduced. Such protection is manifested by a reduction in either the severity or manifestation, even disappearance, of symptoms typically observed in the infected host, a faster recovery time and/or a reduced viral titer in the infected host.

[20] In preferred forms, and especially forms in which the recombinant PCV3 protein is used in an immunogenic composition such as a vaccine, each batch or only individual batches of the assembled PCV3 protein can be tested for inactivation. Thus, another aspect of the present invention relates to an inactivation test for determining the efficiency of inactivation of a recombinant viral vector, comprising the steps of: 1) contacting at least a portion of a culture fluid containing the recombinant viral vector with an inactivating agent, 2) adding a neutralizing agent to neutralization of the inactivating agent and 3) determination of residual infectivity.

[21] In preferred forms, a recombinant viral vector containing PCV3 DNA and expressing PCV3 protein used to infect cells is produced by transfecting into the viral vector a transfer vector that contains the PCV3 gene cloned therein. Preferably, only the portion of the transfer vector that contains the desired PCV3 DNA, such as DNA encoding ORF1, ORF2, or ORF3, is transfected into the viral vector.

[22] The term "transfected into a viral vector" means and is used synonymously with the "introduction" or "cloning" of heterologous DNA into a viral vector, such as, for example, a baculovirus vector. "Transfer vector" means a DNA molecule that includes at least one origin of replication, a heterologous gene, in this case PCV3, DNA sequences that allow the heterologous gene to be cloned into a viral vector. Preferably, the sequences allowing the heterologous gene to be cloned into a viral vector are flanking the heterologous gene. Even more preferably, these flanking sequences are homologous, at least in part, to the sequences of the viral vector. Sequence homology allows further recombination of both molecules, the viral vector and the transfer vector, to produce a recombinant viral vector containing the heterologous gene.

[23] In more preferred forms, the methods of the present invention begin with the isolation of PCV3 DNA. For the purposes of the present invention, any PCV3 gene can be used. PCV3 DNA is preferably amplified by PCR. The resulting DNA is then cloned into a transfer vector.

[24] Thus, in one aspect, the present invention relates to a method for constructing a recombinant viral vector containing PCV3 DNA. This method typically includes the steps of: 1) cloning at least one recombinant PCV3 gene into a transfer vector; and 2) transfecting into a viral vector a portion of a transfer vector containing the recombinant PCV3 gene to obtain a recombinant viral vector. As noted above, the PCV3 gene may encode ORF1, ORF2, or ORF3.

[25] In another aspect, PCV3 DNA can be amplified to step 1) in vitro , where the flanking sequences of the PCV3 DNA are modified. Methods for in vitro amplification of PCV3 DNA and modification of flanking sequences, cloning in vitro amplified PCV3 DNA into a transfer vector and suitable transfer vectors are described above or known to one skilled in the art. Thus, in accordance with another aspect, the present invention relates to a method for constructing a recombinant viral vector containing PCV3 DNA and expressing a desired PCV3 protein, wherein the method includes the steps of: 1) amplifying PCV3 DNA in vitro , where the flanking sequences of the PCV3 DNA are modified, 2) cloning the amplified PCV3 ORF2 DNA into a transfer vector; and 3) transfecting the transfer vector or a portion thereof containing the recombinant PCV3 DNA into a viral vector to generate a recombinant viral vector. In some aspects, modification of the PCV3 DNA flanking sequences is accomplished by introducing a 5'-Kozak sequence and/or an EcoR 1 site.

[26] Another aspect of the present invention relates to a method for producing a composition containing PCV3 protein and an inactivated viral vector. This method typically includes the steps of: 1) cloning the amplified PCV3 DNA into a transfer vector; 2) transfection into the virus of a part of the transfer vector containing recombinant PCV3 DNA; 3) infection of cells in media with a transfected viral vector; 4) expression of a transfected viral vector of a recombinant protein with PCV3 DNA; 5) separation of cells from supernate; 6) extraction of expressed PCV3 protein; and 7) inactivation of the recombinant viral vector. In preferred forms and as described above, the neutralization step, step 8), is performed after step 7). Of course, prior to step 1) PCV3 DNA can be amplified in vitro , preferably with flanking PCV3 DNA sequences as described above.

[27] In another aspect, the present invention relates to a method for preparing a composition, preferably an immunogenic composition, such as a vaccine, for inducing an immune response against PCV3. Typically, this method includes the steps of transfecting a construct into a virus, wherein the construct contains 1) recombinant PCV3 ORF DNA, 2) infection of cells in the growth medium with the transfected virus, 3) ensuring the virus expresses the recombinant PCV3 ORF protein, 4) recovery of the expressed recombinant ORF protein, and 5) preparing the composition by combining the isolated protein with a suitable adjuvant and/or other pharmaceutically acceptable carrier. In some preferred embodiments, the composition also includes at least a portion of a viral vector expressing said PCV3 ORF protein and/or a portion of a cell culture supernatant.

[28] "Adjuvants" used herein may include aluminum hydroxide and aluminum phosphate, for example, saponins Quil A, QS-21 (Cambridge Biotech Inc., Cambridge MA), GPI-0100 (Galenica Pharmaceuticals, Inc., Birmingham , AL), water-in-oil emulsion, oil-in-water emulsion, water-in-oil in water emulsion. The emulsion can be based, in particular, on light liquid paraffin oil (European Pharmacopoeia type); an oil of isoprenoid structure, such as squalene or squalene oil, obtained by oligomerization of alkenes, in particular isobutene or decene; esters of acids or alcohols containing a linear alkyl group, more particularly vegetable oils, ethyl oleate, propylene glycol di-(caprylate/caprate), glyceryl tri-(caprylate/caprate) or propylene glycol dioleate; esters of branched fatty acids or alcohols, in particular esters of isostearic acid. The oil is used in combination with emulsifiers to form an emulsion. Preferred emulsifiers are nonionic surfactants, in particular esters of sorbitan, mannitol (for example, anhydromannitol oleate), glycol, polyglycerol, propylene glycol and oleic, isostearic, ricinoleic or hydroxystearic acid, which are optionally ethoxylated, and polyoxypropylene-polyoxyethylene block copolymers, in particular Pluronic products, in particular L121. See Hunter et al., The Theory and Practical Application of Adjuvants (Ed. Stewart-Tull, D.E.S.). JohnWiley and Sons, NY, pp51-94 (1995) and Todd et al., Vaccine 15:564-570 (1997).

[29] For example, you can use SPT emulsion, described on page 147 of Vaccine Design, The Subunit and Adjuvant Approach, edited by M. Powell and M. Newman, Plenum Press, 1995, and MF59 emulsion, described on page 183 of the same book .

[30] Another example of an adjuvant is a compound selected from polymers of acrylic or methacrylic acid and copolymers of maleic anhydride and an alkenyl derivative. Preferred adjuvant compounds are polymers of acrylic or methacrylic acid, which are cross-linked, in particular with polyalkenyl esters of sugars or polyalcohols. These compounds are known collectively as Carbomer (Phameuropa Vol. 8, No. 2, June 1996). Those skilled in the art may also refer to US Patent No. 2,909,462, which describes such acrylic polymers cross-linked with a polyhydroxylated compound having at least 3 hydroxyl groups, preferably no more than 8, wherein the hydrogen atoms of at least three hydroxyls are replaced by unsaturated aliphatic radicals having at least 2 carbon atoms. Preferred radicals are those containing from 2 to 4 carbon atoms, for example vinyl, allylic and other ethylenically unsaturated groups. Unsaturated radicals may themselves contain other substituents, such as methyl. Particularly suitable are the products sold under the name Carbopol; (BF Goodrich, Ohio, USA). They are cross-linked with allyl sucrose or allyl pentaerythritol. Among them we can mention Carbopol 974P, 934P and 971P; and among the copolymers of maleic anhydride and an alkenyl derivative are EMA copolymers (Monsanto), which are copolymers of maleic anhydride and ethylene. Dissolution of these polymers in water produces an acidic solution which can be neutralized, preferably to physiological pH, to produce an adjuvant solution into which the immunogenic composition or vaccine itself will be incorporated.

[31] Other suitable adjuvants include, but are not limited to, RIBI adjuvant system (Ribi Inc.), block copolymer (CytRx, Atlanta GA), SAF-M (Chiron, Emeryville CA), monophosphoryl lipid A, lipid amine adjuvant Avridin, heat labile E. coli enterotoxin (recombinant or otherwise produced), cholera toxin, IMS 1314 or muramyl dipeptide.

[32] The adjuvant is preferably added in an amount of from about 100 μg to about 10 mg per dose. More preferably, the adjuvant is added in an amount of from about 100 μg to about 10 mg per dose. More preferably, the adjuvant is added in an amount of from about 500 μg to about 5 mg per dose. More preferably, the adjuvant is added in an amount of from about 750 μg to about 2.5 mg per dose. Most preferably, the adjuvant is added in an amount of about 1 mg per dose.

[33] In another aspect of the present invention, a method for preparing an immunogenic composition, such as a vaccine, for inducing an immune response against PCV3 includes the steps of: 1) expressing and extracting the PCV3 ORF protein, and 2) mixing the extracted protein with a suitable adjuvant. Expression step 1) preferably includes the step described for the production and recovery of PCV3 protein. Another optional step of this method includes cloning the amplified PCV3 ORF DNA into a first vector, excising the ORF DNA from the first vector, and using the excised PCV3 ORF DNA for cloning into a transfer vector. The recovery step in this method preferably also includes the step of separating the medium from the cells and cellular debris. This can be accomplished by any conventional method, preferably a method comprising filtering cells, cell debris, and culture media through a filter having a pore size ranging from about 0.45 μm to about 1.0 μm. Finally, for this aspect, the method preferably includes the step of inactivating the virus before incorporating the recovered recombinant PCV3 ORF protein into the composition. If an inactivation step is included, it is preferable to also include a neutralization step as described above.

[34] In addition, the composition may include one or more pharmaceutically acceptable or veterinary acceptable carriers. As used herein, the term "pharmaceutically acceptable carrier" or "veterinarily acceptable carrier" includes any and all solvents, dispersion media, coatings, stabilizing agents, diluents, preservatives, antibacterial and antifungal agents, isotonicity agents, adsorption retardants, etc. d. In one preferred embodiment, the composition provided herein comprises a PCV3 ORF protein isolated from in vitro cultured cells, wherein said cells are infected with a recombinant viral vector containing PCV3 ORF DNA and expressing the PCV3 ORF protein, and wherein the cell culture is treated to inactivate the viral vector, and an equivalent concentration of neutralizing agent is added, and an adjuvant and saline are added. As in other aspects, the PCV3 ORF protein may be ORF1, ORF2, or ORF3. The amount of physiological saline solution, if present, is preferably from about 50 to about 90% (v/v), more preferably from about 60 to 80% (v/v), even more preferably about 70% (v/v). /about.). This method may also optionally include adding a preservative. The protectant used herein refers to an antimicrobial active agent such as, for example, gentamicin, merthiolate and the like. In particular, the addition of a protectant is most preferred to prepare a multiple dose composition. These antimicrobial active agents are added in a concentration effective to prevent any microbiological contamination of the composition of interest or to inhibit the growth of any microbial agent in the composition of interest.

[35] The methods of the present invention may also include the addition of any stabilizing agent, such as, for example, saccharides, trehalose, mannitol, sucrose, and the like, to increase and/or provide shelf life of the product and/or to improve stability.

[36] In another aspect, the present invention relates to products obtained from the methods described above. In particular, the present invention essentially relates to a composition containing a recombinantly expressed PCV3 ORF protein. In some preferred embodiments, the composition substantially also contains an agent suitable for inactivating viral vectors, and contains an agent suitable for inactivating viral vectors. Such products are useful as immunogenic compositions that induce an immune response, and more preferably, provide protective immunity against the onset of clinical signs of PCV3 infection. The composition typically contains a polypeptide or fragment thereof expressed by ORF1, ORF2, ORF3, or any combination of one or more of these PCV3 ORFs as the antigenic component of the composition. Of course, it is understood that the PCF3 ORF polypeptide used in the immunogenic composition in accordance with the present invention can be obtained by any method, including isolation and purification, standard protein synthesis and recombinant methods.

[37] Clinical signs of PCV3 infection are observed both after weaning at 6–8 weeks of age and in growing and lactating pigs at 12–14 weeks of age, and sporadically in other age groups. Clinical signs include the appearance of large, purplish-red, slightly swollen patches of varying sizes and shapes over the chest, abdomen, thighs and forelimbs. Over time, the spots become covered with dark crusts and then disappear, leaving scars. In addition, pigs are depressed, may have a fever, are generally reluctant to move or eat, are losing weight, are panting or in respiratory distress, there may be noticeable swelling or fluid on the limbs and around the eyelids, and superficial lymph nodes may be enlarged, the hair may become rough and the skin pale, they may experience jaundice and some pigs even have diarrhea. Despite the high mortality rate, most pigs with skin lesions die. Signs of PCV3 infection can also be found in the stomach and intestines, such as stomach ulcers and hemorrhage, fluid in the abdominal cavity; lesions can be found in the lungs, tonsils, spleen, liver and kidneys, which are enlarged in size and become pale and mottled with numerous small hemorrhages appearing on the surface.

[38] Any PCV3 ORF would be effective as a source of PCP3 ORF DNA and/or polypeptide used herein. In preferred forms, the DNA ORF is ORF2. The preferred PCV3 ORF2 protein is SEQ ID No. 6, but those skilled in the art will appreciate that this sequence, as well as the sequence for ORF1 and ORF3, may differ by up to 10% with sequence homology but still retain antigenic characteristics, which make it useful for use in immunogenic compositions. The antigenic characteristics of the immunological composition can, for example, be assessed by experimentation. Moreover, the antigenic characteristic of the modified antigen is still retained if the modified antigen provides at least 70%, preferably 80%, more preferably 90% of protective immunity compared to the PCV3 ORF protein encoded by the polynucleotide sequence of SEQ ID No. 3, 5 or 7. In some forms, immunogenic portions of the PCV3 ORF protein are used as the antigenic component in the composition. As used herein, the term “immunogenic moiety” refers to truncated and/or substituted forms or fragments of the PCV3 ORF2 protein and/or polynucleotide, respectively. Preferably, such truncated and/or substituted forms or fragments may contain at least 6 contiguous amino acids from the full-length ORF polypeptide. More preferably, the truncated or substituted forms or fragments may have at least 10, more preferably at least 15, and even more preferably at least 19 contiguous amino acids from the full-length ORF polypeptide. In addition, it is understood that such sequences may be part of larger fragments or truncated forms. Preferably, such truncated or substituted forms or fragments may contain at least 18 contiguous nucleotides from the full-length ORF nucleotide sequence, for example, SEQ ID No. 3, 5 or 7. More preferably, the truncated or substituted forms or fragments may have at least 30 or more preferably at least 45 and even more preferably at least 57 contiguous nucleotides of the full-length ORF nucleotide sequence.

[39] “Sequence identity”, as is known in the art, refers to the relationship between two or more polypeptide sequences or two or more polynucleotide sequences, namely between a reference sequence and a given sequence compared to a reference sequence. Sequence identity is determined by comparing a given sequence to a reference sequence following an optimal sequence alignment that produces the highest degree of sequence similarity, as determined by matches between strands of such sequences. In this alignment, sequence identity is established by positional comparison, for example, sequences are “identical” at a particular position if the nucleotides or amino acid residues at that position are identical. The total number of such positional identities is then divided by the total number of nucleotides or residues in the reference sequence, yielding the percentage of sequence identity. Sequence identities can be readily calculated by known methods, including, but not limited to, those described in Computational Molecular Biology, Lesk, A. N., ed., Oxford University Press, New York (1988), Biocomputing: Informatics and Genome Projects, Smith, D. W., ed., Academic Press, New York (1993); Computer Analysis of Sequence Data, Part I, Griffin, A. M., and Griffin, H. G., eds., Humana Press, New Jersey (1994); Sequence Analysis in Molecular Biology, von Heinge, G., Academic Press (1987); Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds., M. Stockton Press, New York (1991); and Carillo, H., and Lipman, D., SIAM J. Applied Math., 48: 1073 (1988), the contents of which are incorporated herein by reference. The preferred methods for determining sequence identity are designed to determine the closest match between the sequences being tested. Methods for determining sequence identity are implemented in publicly available computer programs that determine the identity between given sequences. Examples of such programs include, but are not limited to, the GCG software package (Devereux, J., et al., Nucleic Acids Research, 12(1):387 (1984)), BLASTP, BLASTN, and FASTA (Altschul, S. F. et al., J Molec Biol 215:403-410 (1990) The BLASTX program is publicly available from NCBI and other sources (BLAST Manual, Altschul, S. et al., NCVI NLM NIH Bethesda, MD 20894, Altschul, S. F. et al. al., J. Molec. Biol., 215:403-410 (1990), the contents of which are incorporated herein by reference.Sequence alignment using these programs is accomplished by assigning default gap opening penalties to obtain highest level of sequence identity between a given and a reference sequence. By way of illustration, a polynucleotide with a nucleotide sequence having at least 85%, preferably 90%, more preferably 95% "sequence identity" with a reference nucleotide sequence means that the nucleotide sequence of the given polynucleotide identical to the reference sequence, except that a given polynucleotide sequence may include up to 15, preferably up to 10, even more preferably up to 5 point mutations for every 100 nucleotides of the reference nucleotide sequence. In other words, in a polynucleotide having a nucleotide sequence having at least 85%, preferably 90%, more preferably 95% identity with a reference nucleotide sequence, up to 15%, preferably 10%, more preferably 5% of the total number of nucleotides in the reference sequence may be removed or replaced by another nucleotide, or up to 15%, preferably 10%, more preferably 5% of the total nucleotides in the reference sequence may be inserted into the reference sequence. These reference sequence mutations may occur at the 5' or 3' terminal positions of the reference nucleotide sequence or anywhere between these terminal positions, either individually among the nucleotides of the reference sequence or as one or more contiguous groups within the reference sequence. Similarly, a polypeptide with a given amino acid sequence having at least 85%, preferably 90%, more preferably 95% sequence identity with a reference amino acid sequence means that the amino acid sequence of the polypeptide is identical to the reference sequence, except that the polypeptide sequence may include up to 15, preferably up to 10, more preferably up to 5 amino acid changes for every 100 amino acids of the reference amino acid sequence. In other words, to obtain a given polypeptide sequence having at least 85%, preferably 90%, more preferably 95% sequence identity with a reference amino acid sequence, up to 15%, preferably up to 10%, even more preferably up to 5% amino acid residues in the reference sequences may be deleted or replaced by another amino acid, or multiple amino acids in an amount of up to 15%, preferably up to 10%, more preferably up to 5% of the total amino acid residues in the reference sequence may be inserted into the reference sequence. These reference sequence changes may occur at the amino- or carboxy-terminal positions of the reference amino acid sequence or anywhere between these terminal positions, either individually among residues of the reference sequence or as one or more contiguous groups within the reference sequence. Preferably, positions of residues that are not identical are distinguished by conservative amino acid substitutions. However, when determining sequence identity, conservative substitutions are not considered a match.

[40] "Sequence homology" as used herein refers to a method for determining the relatedness of two sequences. To determine sequence homology, two or more sequences are optimally aligned and breaks are introduced where necessary. However, unlike "sequence identity", when determining sequence homology, conservative amino acid substitutions are considered as matches. In other words, to obtain a polypeptide or polynucleotide having 95% sequence homology with a reference sequence, 85%, preferably 90%, even more preferably 95% of the amino acid residues or nucleotides in the reference sequence must match or contain a conservative substitution for another amino acid or nucleotide, or amino acids or nucleotides may be inserted into the reference sequence in amounts up to 15%, preferably up to 10%, more preferably up to 5% of the total amino acid residues or nucleotides in the reference sequence, not including conservative substitutions. Preferably, the homologous sequence contains at least a region of 50, more preferably 100, even more preferably 250, even more preferably 500 nucleotides.

[41] "Conservative substitution" refers to the replacement of an amino acid residue or nucleotide with another amino acid residue or nucleotide having similar characteristics or properties, including size, hydrophobicity, etc., such that the overall functionality is not significantly changed.

[42] “Isolated” means altered “by the actions of man” in relation to a state of nature, i.e., if something occurs in nature, it has been altered or removed from its original environment, or both. For example, a polynucleotide or polypeptide naturally present in a living organism is not “isolated,” but the same polynucleotide or polypeptide, separated from materials coexisting with it in its natural state, is “isolated,” as that term is used herein .

[43] In a further aspect, the present invention provides an immunogenic composition effective for reducing the severity of clinical symptoms associated with PCV3 infection containing the PCF3 ORF protein. Preferably, the PCV3 ORF2 protein is selected from the group consisting of: 1) a polypeptide containing the sequence of SEQ ID No. 4, 6, 8, or any combination thereof; 2) any polypeptide that is at least 90% homologous to polypeptide 1); 3) any immunogenic part of polypeptides 1) and/or 2); 4) an immunogenic part 3) containing at least 10 contiguous amino acids included in the sequence SEQ ID No. 4, 6 or 8; 5) a polypeptide equivalent (due to the degeneracy of the genetic code) to the polypeptide encoded by DNA containing the sequence SEQ ID No. 3, 5 or 7; 6) any polypeptide encoded by a polynucleotide that is at least 80% homologous to polynucleotide 5); 7) any immunogenic part of the polypeptides encoded by polynucleotide 5) and/or 6); or 8) an immunogenic portion 7), wherein the polynucleotide encoding said immunogenic portion contains at least 30 contiguous nucleotides included in SEQ ID No. 3, 5, or 7.

[44] In preferred forms, these immunogenic portions may have the immunogenic characteristics of the PCV3 ORF protein, which is encoded by SEQ ID No. 3, 5 or 7.

[45] In yet another aspect, the PCF3 ORF2 protein is present in the immunological composition at a level of antigen incorporation effective to induce a desired immune response, namely, to reduce the incidence or severity of clinical signs resulting from PCV3 infection. Preferably, the level of PCV3 ORF2 protein incorporation is at least 0.2 μg of antigen/ml of finished immunogenic composition (μg/ml), more preferably from about 0.2 to about 400 μg/ml.

[46] The polypeptide is included in a composition that can be administered to an animal susceptible to PCV3 infection. In preferred forms, the composition may also include additional components known to those skilled in the art (see also Remington's Pharmaceutical Sciences. (1990). 18th ed. Mack Publ., Easton).

[47] One skilled in the art will appreciate that the composition of the present application may include known injectable, physiologically acceptable sterile solutions. To prepare a ready-to-use solution for parenteral injection or infusion, ready-made aqueous isotonic solutions, such as, for example, saline or appropriate solutions of plasma proteins, can be used. In addition, the immunogenic and vaccine compositions of the present invention may include diluents, isotonicity agents, stabilizers or adjuvants. Diluents may include water, saline, dextrose, ethanol, glycerin, and the like. Isotonicity maintaining agents may include sodium chloride, dextrose, mannitol, sorbitol and lactose, among others. Stabilizers include, but are not limited to, albumin and alkali metal salts of ethylenediaminetetraacetic acid. Suitable adjuvants are those described above.

[48] In yet another aspect, the immunogenic composition of the present invention further comprises a pharmaceutically acceptable salt, preferably a phosphate salt, in physiologically acceptable concentrations. Preferably, the pH of said immunogenic composition is adjusted to physiological pH, which means about 6.5 to 7.5.

[49] The immunogenic compositions described herein may further include one or more other immunomodulatory agents, such as, for example, interleukins, interferons, or other cytokines. Immunogenic compositions may also include gentamicin and merthiolate. In another preferred embodiment, the present invention provides vaccine compositions containing from about 1 μg to about 60 μg/ml of antibiotics, and more preferably less than about 30 μg/ml of antibiotics.

[50] Immunogenic compositions containing recombinant PCV3 ORF protein as provided herein have been found to be very effective in reducing the severity or incidence of clinical signs associated with PCV3 infections, even preventing the occurrence of such signs.

[51] Another aspect of the present invention relates to a kit. Typically, the kit includes a container containing at least one dose of an immunogenic PCV3 ORF protein composition as defined herein, wherein one dose contains at least 2 μg of PCF3 ORF protein. Said container may contain from 1 to 250 doses of the immunogenic composition. In some preferred forms, the container contains 1, 10, 25, 50, 100, 150, 200, or 250 doses of an immunogenic PCV3 ORF protein composition. Preferably, each of the containers containing more than one dose of the immunogenic PCV3 ORF protein composition further contains an antimicrobial active agent. These agents are, for example, antibiotics including gentamicin and merthiolate and the like. Thus, one aspect of the present invention relates to a container that contains from 1 to 250 doses of an immunogenic PCV3 ORF protein composition, with one dose containing at least 2 μg of PCV3 ORF protein and gentamicin and/or merthiolate, preferably from about 1 μg/ ml to about 60 μg/ml of antibiotics, and more preferably less than about 30 μg/ml. In preferred forms, the kit also includes instructions for use containing information for intramuscular administration of at least one dose of an immunogenic PCV3 ORF protein composition to animals, preferably pigs and piglets, to reduce the frequency and/or severity of clinical symptoms associated with PCV3 infection. Moreover, in accordance with yet another aspect, said instructions for use comprise information about a second or subsequent administration(s) of at least one dose of the ORF2 PCF3 immunogenic composition, wherein the second administration or any subsequent administration occurs at least 14 days after the initial or any previous administration. In some preferred forms, said instructions also include information about administering the immune stimulant. Preferably, said immune stimulant should be administered at least twice. Preferably at least 3, more preferably at least 5 and even more preferably at least 7 days elapse between the first and second or any subsequent administration of the immunostimulant. Preferably, the immune stimulant is administered at least 10 days, preferably 15, even more preferably 20, and even more preferably at least 22 days after the initial administration of the immunogenic PCV3 ORF protein composition. It is understood that any immune stimulant known to one skilled in the art may also be used. "Immune stimulant" as used herein means any agent or composition that can induce a general immune response, preferably without inducing or increasing a specific immune response, such as an immune response to a particular pathogen. According to the instructions, it is also suggested to administer an immune stimulant in a suitable dose. The kit may also contain a second container including at least one dose of an immune stimulant.

[52] Another aspect of the present invention relates to a kit as described above containing a PCV3 ORF immunogenic composition as provided by the present invention and instructions for use, wherein the instructions for use further include information that the PCV3 ORV immunogenic composition is to be co-administered , or at about the same time as the immunogenic composition containing an additional antigen effective to reduce the severity or frequency of clinical signs associated with another swine pathogen. Preferably, the instructions contain information about when to administer the composition containing the PCV3 ORF and the immunogenic composition containing the additional antigen.

[53] Another aspect relates to the use of any of the compositions presented herein as a medicinal product, preferably as a veterinary medicinal product, even more preferably as a vaccine. In addition, the present invention also relates to the use of any of the compositions described herein for the preparation of a medicament for reducing the severity of clinical symptoms associated with PCV3 infection. Preferably, the medicinal product is intended to prevent PCV3 infection in pigs, even more preferably piglets.

[54] Another aspect relates to a method of (1) preventing PCV3 infection or re-infection or (2) reducing the incidence or severity of or eliminating clinical symptoms caused by PCV3 in a patient, comprising administering any of the immunogenic compositions provided herein to the patient. Preferably, the patient is a pig. It is clear that the reduction occurs compared to a patient who did not receive the composition of the present invention. Preferably, one or two doses of the immunogenic composition are administered, with one dose preferably containing at least about 2 μg of PCV3 ORF protein. Another aspect relates to the above-described method of treatment comprising a second administration of an immunogenic composition. Preferably, the second administration is performed using the same immunogenic composition, preferably with the same amount of PCV3 ORF protein. Preferably, the second administration is performed at least 14 days after the initial administration, even more preferably at least 4 weeks after the initial administration. In preferred forms, the method is effective immediately after administration of one dose of the immunogenic composition, without requiring a second or subsequent administration to provide protection to the patient.

[55] In yet another aspect, the present invention provides a multivalent combination vaccine that includes an immunological agent effective to reduce the incidence or severity of PCV3 infection and at least one immunologically active component directed against another organism that causes disease in pigs.

[56] In particular, an immunological agent effective in reducing the frequency or severity of PCV3 infection is PCV3 antigen. Preferably, said PCV3 antigen is a PCV3 ORF protein as provided herein, or any immunogenic composition described above that contains a PCV3 ORF protein. The PCV3 ORF protein may be ORF1, ORF2, ORF3, or any combination thereof. For example, ORF1 in combination with ORF2, ORF2 in combination with ORF3, ORF1 in combination with ORF3, and the combination of ORF1, 2 and 3 are possible combinations of PCV3 ORFs that can be combined in one immunogenic composition. In preferred forms, the ORF is ORF2 or a combination including ORF2.

[57] The terms “immunogenic protein,” “immunogenic polypeptide,” or “immunogenic amino acid sequence,” as used herein, refer to any amino acid sequence that elicits an immune response in a host against a pathogen containing said immunogenic protein, immunogenic polypeptide, or immunogenic amino acid sequence. "Immunogenic protein", "immunogenic polypeptide" or "immunogenic amino acid sequence" as used herein includes the full-length sequence of any proteins, analogs thereof, or immunogenic fragments thereof. By "immunogenic fragment" is meant a protein fragment that includes one or more epitopes and thus induces an immune response to the corresponding pathogen. Such fragments can be identified by any number of epitope mapping techniques well known in the art. See, for example, protocols for epitope mapping in Methods in Molecular Biology, Vol. 66 (Glenn E. Morris, Ed., 1996) Humana Press, Totowa, New Jersey. For example, linear epitopes can be determined, for example, by simultaneously synthesizing large numbers of peptides on solid supports, peptides corresponding to parts of the protein molecule, and by reacting the peptides with antibodies while the peptides are still attached to the supports. Such methods are known in the art and are described, for example, in US Pat. No. 4,708,871; Geysen et al. (1984) Proc. Natl. Acad. Sci. USA 81:3998-4002; Geysen et al. (1986) Molec. Immunol. 23:709-715. Likewise, conformational epitopes can be easily identified by determining the spatial conformation of amino acids, for example, by X-ray crystallography and two-dimensional nuclear magnetic resonance. See, for example, epitope mapping protocols above. This definition also includes synthetic antigens, such as polyepitopes, flanking epitopes, and other recombinant or synthetically produced antigens. See, for example, Bergmann et al. (1993) Eur. J. Immunol. 23:2777-2781; Bergmann et al. (1996), J. Immunol. 157:3242-3249; Suhrbier, A. (1997), Immunol. and Cell Biol. 75:402-408; Gardner et al., (1998) 12th World AIDS Conference, Geneva, Switzerland, June 28-July 3, 1998.

[58] The other disease-causing organism in pigs is preferably selected from the group consisting of:Actinobacillus pleuropneumonia; adenovirus; an alphavirus such as eastern equine encephalomyelitis viruses;Bordetella bronchiseptica;Brachyspira spp.., preferablyB. hyodyenteriae; B. piosicoli, Brucella suis, preferably biovars 1, 2 and 3; classical swine fever virus;Clostridium spp.., preferablyCl. difficile , Cl. perfringens types A, B and C,Cl. novyi, Cl.septicum, Cl. tetani; coronavirus, preferably porcine respiratory coronavirus;Eperythrozoonosis suis; Erysipelothrix rhsiopathiae; Escherichia coli; Haemophilus parasuis, preferably subtypes 1, 7 and 14; hemagglutinating encephalomyelitis virus; Japanese encephalitis virus;Lawsonia intracellularis; Leptospira spp..;Leptospira australis; Leptospira canicola; Leptospira grippotyphosa; Leptospira icterohaemorrhagicae; AndLeptospira interrogans; Leptospira pomona; Leptospira tarassovi; Mycobacterium spp.. preferablyM. avium; M. intracellulare; AndM.bovis ; Mycoplasma hyopneumoniae (Mhyo); Pasteurella multocida; porcine cytomegalovirus; porcine parvovirus; porcine reproductive and respiratory syndrome (PRRS) virus; pseudoreceptor virus; rotavirus;Salmonella spp..; preferablyS. thyhimurium; AndS. choleraesuis ; Staph. hyicus; Staphylococcus spp.. preferablyStreptococcus spp.., preferably Strep. suis; porcine herpes virus; swine flu virus; swinepox virus; swinepox virus; vesicular stomatitis virus; porcine vesicular exanthema virus;Leptospira Hardjo; and/orMycoplasma hyosynoviae.

[59] As used herein, "immunologically active component" means a component that induces or stimulates an immune response in an animal to which the component is administered. According to a preferred embodiment, said immune response is directed to said component or to a microorganism containing said component. According to another preferred embodiment, the immunological active component is an attenuated microorganism including a modified live virus (MLV), a killed microorganism or at least an immunological active part of a microorganism.

[60] "Immunologically active part of a microorganism" as used herein means a protein, sugar and/or glycoprotein-containing fraction of a microorganism that contains at least one antigen that induces or stimulates an immune response in the animal to which the component is administered. According to a preferred embodiment, said immune response is directed to said immunologically active part of a microorganism or a microorganism containing said immunologically active part.

[61] In another aspect, the present invention relates to infectious chimeric DNA clones of porcine circovirus (PCV3) and live chimeric viruses derived from the DNA clones, which can be used as vaccines. New live chimeric, genetically avirulent viruses are derived from the non-pathogenic PCV1 genomic structure in which the immunogenic gene of the pathogenic PCV3 strain is replaced by a gene from PCV1, usually at the same corresponding position. Also included within the scope of the invention are biologically functional plasmids, viral vectors, and the like, which contain the novel recombinant nucleic acid molecules described herein, suitable host cells transfected with vectors containing this DNA and immunogenic polypeptide expression products. The present invention includes a new method for protecting pigs or providing protection against viral infection or the onset of clinical signs described above, as well as against post-weaning multisystem wasting syndrome (PMWS), pneumonia, reproductive failure and porcine dermatitis and nephropathy syndrome (PDNS), caused by PCV3, wherein the method comprises administering to a pig in need of such protection an immunologically effective amount of a vaccine containing, for example, chimeric DNA cloned into a plasmid, a chimeric virus derived from a chimeric DNA clone, polypeptide products expressed from the DNA described herein, or any combination of them. Another aspect of the invention relates to new mutants of the immunogenic capsid gene and PCV3 protein and the introduction of mutations into chimeric clones to facilitate cell culture growth and ensure vaccine safety. In another aspect, novel infectious PCV3 DNA molecules and reciprocal chimeric PCV DNA clones are provided that are useful as experimental models for the production or characterization of new avirulent viral vaccines.

[62] In one aspect, the present invention relates to infectious molecular and chimeric nucleic acid molecules of porcine circovirus (PCV), live chimeric viruses derived from the chimeric nucleic acid molecule, and veterinary vaccines for protecting pigs against viral infection, pneumonia, reproductive deficiency, PMWS and/or PDNS caused by PCV3. The invention also includes immunogenic polypeptide expression products that can be used as vaccines. The novel avirulent, infectious chimeric PCV DNA molecule (PCV1-3) contains in the PCV1 genome a nucleic acid molecule encoding infectious, non-pathogenic PCV1 that contains the immunogenic open reading frame (ORF) gene of pathogenic PCV3 in place of the PCV1 ORF gene. The infectious chimeric PCV1-3 DNA clone preferably contains the immunogenic capsid gene (ORF2) of PCV3 DNA cloned into the genomic backbone of the infectious, non-pathogenic PCV1 DNA clone. Typically, the capsid gene of PCV3 DNA replaces the ORF2 gene with PCV1 DNA in the non-pathogenic PCV1 genomic structure, but it is believed that a variety of positional permutations can be created through genetic engineering to produce other avirulent or attenuated chimeric DNA clones. Disclosed is a reciprocal chimeric infectious DNA clone PCV3-1 between PCV1 and PCV3, used as a control for the assay of the chimeric PCV1-3 clone of the invention, which is constructed by replacing the PCV3 capsid gene with the PCV1 gene in the backbone of the pathogenic infectious DNA clone PCV3. In addition to the experimental model, the reciprocal chimeric DNA clone PCV3-1 may find application in the manufacture of tailored vaccines.

[63] In other forms, PCV3 ORF3 may be replaced by ORF2 or ORF3 of the infectious clone PCV1-3 or PCV3-1. In other preferred forms, PCV3 ORF1 and ORF3 may be replaced by PCV3 ORF2 of the infectious clone PCV1-3 or PCV3-1.

[64] It is shown below that the cloned PCV3 genomic DNA described herein is in vitro and in vivo infectious when transfected into PK-15 cells and is transfected into pigs. Infectious PCV3 DNA clones cause the pathological lesions characteristic of PMWS and PDNS in pigs, leading to improved clinical disease characterization and understanding of viral spread in tissue cells. This novel, easily reproducible pathogenic agent is suitable for use in developing a suitable vaccination program to prevent PMWS and PDNS in pigs.

[65] Pigs can also be infected with the new chimeric DNA clone PCV1-3 either by in vitro transfection of PK-15 cells or by in vivo administration. In transfected PK-15 cells, one preferential chimeric DNA clone PCV1-3 can express the PCV3 ORF2 capsid antigen (PCV3 immunogenic capsid protein), while the reciprocal chimeric DNA clone PCV3-1 can express the PCV1 capsid antigen, which can be demonstrated by immunofluorescence assay (IFA) using antibodies specific for PCV1 or PCV3 capsid antigen. Seroconversion to PCV3-specific antibody will be detected in pigs inoculated with the infectious PCV3 clone as well as the chimeric PCV1-3 clone. The detection of seroconversion to PCV3-specific antibody establishes that the chimeric DNA clone PCV1-3 induces PCV3-specific antibody in infected pigs and therefore protects inoculated pigs from PCV3 infection.

[66] Surprisingly, it was discovered that a chimeric infectious DNA clone of PCV1-3, having the immunogenic capsid gene (ORF2) of pathogenic PCV3 cloned into the non-pathogenic genomic backbone of PCV1, can induce a specific antibody response to the pathogenic capsid antigen of PCV3, which is clearly exclusively non-pathogenic character of PCV1 in pigs. Animals inoculated with a chimeric infectious DNA clone of PCV1-3 can develop a mild infection similar to that of animals inoculated with PCV1 by seroconverting to antibody to the ORF2 capsid protein of the pathogenic PCV3. The average duration of viremia observed in animals inoculated with PCV1 and chimeric PCV1-3 will be shorter than in animals inoculated with pathogenic PCV3. The absence of detectable PCV1-3 chimeric viremia in some inoculated animals will not affect seroconversion to antibody to the PCV3 ORF2 capsid protein in PCV1-3-inoculated pigs. According to the results obtained, despite the short and undetectable viremia of chimeric PCV1-3 in some inoculated animals, the chimeric PCV1-3 virus is able to induce an antibody response to the capsid protein ORF2 of PCV3. The distinct ability of the chimeric infectious DNA clone PCV1-3 to induce an immune response specific to the pathogenic immunogenic capsid protein ORF2 of PCV3, while still non-pathogenic in pigs, makes the chimeric clone(s) PCV1-3 particularly useful as an engineered live attenuated vaccine and other types of vaccines. Suitable cells containing the chimeric nucleic acid molecule will uniquely produce live, infectious porcine circoviruses. In some preferred forms, a live, infectious chimeric virus is produced from a chimeric DNA clone by in vitro and in vivo transfection of PK-15 cells. The invention also provides for the production of a chimeric virus from complementary strand or nucleotide sequences having high homology of at least 90%, more preferably 91, 92, 93, 94, 95, 96, 97, 98 or 99% homology with the chimeric nucleotide sequence.

[67] Also included within the scope of the present invention are biologically functional plasmids, viral vectors, etc., which contain the novel recombinant nucleic acid molecules described herein, suitable host cells transfected with vectors containing chimeric and molecular DNA clones and immunogenic polypeptide expression products. Some particularly preferred immunogenic proteins may have the amino acid sequence set forth in SEQ ID No. 4, 6 or 8. Biologically active variants thereof are also included within the scope of the invention. One skilled in the art will know how to modify, replace, remove, etc. amino acid(s) from a polypeptide sequence and produce biologically active variants that retain the same or substantially the same activity as the parent sequence without undue effort.

[68] To obtain the immunogenic polypeptide products of the present invention, the method may include the following steps: growing, under suitable nutritional conditions, prokaryotic or eukaryotic host cells transfected with the novel recombinant nucleic acid molecules described herein in a manner allowing expression of said polypeptide products, and isolating the required polypeptide expression products of said nucleic acid molecules by standard methods known in the art. It is contemplated that immunogenic proteins can be produced by other methods, such as, for example, biochemical synthesis and the like.

[69] Another embodiment of the present invention relates to novel mutations in the nucleotide and amino acid sequences of the immunogenic capsid gene and protein of PCV3. PCV3 is the causative agent of PMWS, PDNS, pneumonia, and reproductive failure, whereas porcine circovirus type I (PCV1), derived from PK-15 cell culture, is nonpathogenic in pigs. A PCV1-3 vaccine containing the mutation can be further tested in cell culture using routine procedures to select a combination that promotes cell culture growth or provides greater safety in pig vaccination by further reducing the virulence properties of PCV3 if they persist. Despite the advantage of the PCV1-3 chimera in its natural avirulence feature, the alternative use of a mutated PCV3 ORF2 to produce a PCV1-3 chimera provides another embodiment of the present invention that is available if further enhancement of the safety of a natural live chimera vaccine is warranted.

[70] Chimeric viral and molecular DNA clone vaccines and methods of their use are also included within the scope of this application. Inoculated pigs are protected from severe viral infection, pneumonia, reproductive failure, PDNS and PMWS caused by PCV3. The novel method protects pigs requiring protection from viral infection, pneumonia, reproductive failure, PDNS or PMWS by administering to the pigs an immunologically effective amount of the vaccine of the present application, such as, for example, a vaccine containing an immunogenic amount of chimeric PCV1-3 DNA, a cloned chimeric a virus, plasmid or viral vector containing chimeric PCV1-3 DNA, polypeptide expression products, recombinant PCV3 DNA, etc. Other antigens, such as those described above, and immune stimulants may be administered simultaneously to pigs to provide broad spectrum protection against infections. Such simultaneous administration may be performed as separate administrations or combined as a single multivalent composition.

[71] The vaccines contain, for example, infectious PCV1-3 chimeric DNA, cloned PCV chimeric DNA genome into suitable plasmids or vectors, such as, for example, pSK vector, avirulent live chimeric virus, inactivated chimeric virus, etc., in combinations with a non-toxic, physiologically acceptable carrier and, optionally, one or more adjuvants as described above. The vaccine may also contain the infectious molecular DNA clone PCV3 described herein. The avirulent live virus vaccine of the present invention provides an advantage over traditional virus vaccines that use either attenuated live viruses that are at risk of reverting to a virulent state, or a whole killed virus that has been propagated in cell culture and that cannot induce sufficient immune response. antibody response to protect against viral disease.

[72] Although the live virus vaccine has some advantages, other types of vaccines can be used to inoculate pigs with the new chimeric virus and the other antigens described herein. The production of inactivated viral vaccines, for example propagation of the virus from an infectious DNA clone, is carried out by methods known in the art or described in this application. Inactivation of the serial virus is then optimized according to conventional protocols known to those skilled in the art.

[73] Inactivated viral vaccines can be prepared by treating isolated virus or chimeric virus obtained from cloned PCV DNA with inactivating agents such as formaldehyde or hydrophobic solvents, acids, etc., irradiation with ultraviolet light or x-rays, heat and etc. Inactivation is performed in a manner conventional in the art. A virus is considered inactivated if it is unable to infect a cell susceptible to infection.

[74] To obtain an attenuated vaccine from pathogenic clones, live pathogenic tissue culture-adapted PCV3 is first attenuated (rendered non-pathogenic or harmless) by methods known in the art, typically through serial passage through cell cultures. Attenuated pathogenic clones can also be obtained by gene deletion or mutation of virus-producing genes. Attenuated PCV3 viruses can then be used to create additional chimeric PCV1-3 viruses that retain the non-pathogenic phenotype of PCV1 but may differ in the expression of immunogenicity characteristics selected from the PCV3 genome using recombinant technology.

[75] The present invention also provides vaccines comprising the nucleotide sequence of the PCV3 genome, SEQ ID No. 1, SEQ ID No. 3, SEQ ID No. 5, SEQ ID No. 7, or a homolog or fragment thereof and pharmaceutical or veterinary acceptable carrier. In one embodiment of the invention, the nucleotide sequence is selected from a homologue or fragment thereof. In another embodiment, the homolog has at least 90% sequence identity to SEQ ID No. 2, SEQ ID No. 4, SEQ ID No. 6, or SEQ ID No. 8. In another embodiment, the vaccine further comprises an adjuvant.

[76] Another aspect of the present invention relates to vaccines comprising a vector and an acceptable pharmaceutical or veterinary carrier, the vector comprising a PCV3 genome nucleotide sequence such as SEQ ID Nos. 2, 4, 6, 8 or any combination thereof, or a homologue thereof or fragment.

[77] The present invention also relates to vaccines, immunogenic compositions containing a cell and an acceptable pharmaceutical or veterinary carrier, where the cell is transformed with the nucleotide sequence of the PCV3 genome or a homologue or fragment thereof.

[78] In addition, the present invention relates to vaccines or immunogenic compositions containing a pharmaceutically acceptable carrier and one polypeptide, wherein said one polypeptide consists of SEQ ID No. 2, 4, 6 or 8.

[79] In addition, the present invention provides methods for immunizing a mammal against PCV3, comprising administering to the mammal an effective amount of the vaccine or immunogenic composition described above.

[80] The present invention relates to nucleotide sequences of the PCV3 genome selected from the sequences of SEQ ID No. 1, SEQ ID No. 3, SEQ ID No. 5, SEQ ID No. 7 or one of their fragments.

[81] The present invention also provides nucleotide sequences characterized in that they are selected from: a) the nucleotide sequence of a specific fragment of SEQ ID No. 1, SEQ ID No. 3, SEQ ID No. 5, SEQ ID No. 7, or one of their fragments; b) a nucleotide sequence homologous to the nucleotide sequence such as defined in a); c) a nucleotide sequence complementary to a nucleotide sequence such as those defined in a) or b), and the nucleotide sequence of their corresponding RNA; d) a nucleotide sequence capable of hybridizing under stringent conditions with a sequence such as defined in a), b) or c); e) a nucleotide sequence comprising a sequence such as defined in a), b), c) or d); and f) a nucleotide sequence modified by a nucleotide sequence such as defined in a), b), c), d) or e).

[82] In the context of the present invention, a nucleotide, polynucleotide or nucleic acid sequence means both double-stranded and single-stranded DNA in monomeric and dimeric (so-called tandem) forms, and the transcription products of said DNA.

[83] It should be understood that the present invention does not relate to genomic nucleotide sequences taken in their natural environment, i.e. in a natural state. It refers to sequences that can be isolated, purified or partially purified, starting with separation methods such as, for example, ion exchange chromatography, by exclusion based on molecular size or affinity, or alternative fractionation methods based on solubility in various solvents, or starting from genetic engineering methods such as amplification, cloning and subcloning, and the sequences of the invention can be transferred using vectors.

[84] By a nucleotide sequence complementary to the sequence according to the invention, it should be understood any DNA of reverse orientation, the nucleotides of which are complementary to the nucleotides of the sequence according to the invention (antiparallel sequence).

[85] Hybridization under stringent conditions with a nucleotide sequence according to the invention means hybridization under conditions of temperature and ionic strength selected to ensure hybridization between two fragments of complementary DNA.

[86] Among the nucleotide sequences of the invention, preferred are nucleotide sequences that can be used as a primer or probe in methods that allow the production of homologous sequences of the invention, these methods such as polymerase chain reaction (PCR), cloning and nucleic acid sequencing, well known to those skilled in the art.

[87] Among these nucleotide sequences according to the invention, also preferred are sequences that can be used as a primer or probe in methods that allow the diagnosis of the presence of PCV3 or one of its variants, such as those defined below.

[88] Also preferred are nucleotide sequences of the invention capable of modulating, inhibiting or inducing PCV3 gene expression and/or capable of modulating the PCV3 replication cycle in a host cell and/or in a host organism. The replication cycle means the invasion and propagation of PCV3 and its transmission from host cell to host cell in the host.

[89] Among the mentioned nucleotide sequences according to the invention are disclosed sequences that correspond to open reading frames, called ORF sequences, and which encode polypeptides, such as, for example, the sequences of SEQ ID No. 4 (ORF1), SEQ ID No. 6 (ORF2) and SEQ ID No. 8 (ORF3), respectively. Fragments of the nucleotide sequence according to the invention can be obtained, for example, by specific amplification, such as PCR, or by digestion of the nucleotide sequences according to the invention with appropriate restriction enzymes, these methods are particularly described in Sambrook et al., 1989. These are representative fragments can also be obtained by chemical synthesis, if they are not very large in size, and by methods well known to those skilled in the art.

[90] Modified nucleotide sequence means any nucleotide sequence obtained by mutagenesis by methods well known to one skilled in the art and containing modifications relative to the normal sequences of the invention, for example mutations in the regulatory and/or promoter sequences of expression of the polypeptide, in particular leading to a change in the rate expression of the specified polypeptide or to modulate the replication cycle.

[91] Modified nucleotide sequence also means any nucleotide sequence encoding a modified polypeptide, such as defined below.

[92] The present invention relates to PCV3 nucleotide sequences of the invention, characterized in that they are selected from the sequences of SEQ ID No.1, SEQ ID No.2, SEQ ID No.3, SEQ ID No.4, SEQ ID No.5 , SEQ ID No.6, SEQ ID No.7, SEQ ID No.8 or one of their fragments.

[93] The invention also relates to nucleotide sequences, characterized in that they contain a nucleotide sequence selected from: a) a nucleotide sequence of SEQ ID No.1, SEQ ID No.3, SEQ ID No.5, SEQ ID No.7, SEQ ID No.9 or one of its fragments; b) the nucleotide sequence of a specified sequence fragment, such as defined in a); c) a homologous nucleotide sequence having at least 80% identity with the sequence such as defined in a) or b); d) a complementary nucleotide sequence or RNA sequence corresponding to the sequence such as defined in a), b) or c); and e) a nucleotide sequence modified by a sequence such as those defined in paragraphs a), b), c) or d).

[94] With respect to homology to the nucleotide sequences of SEQ ID No. 1, SEQ ID No. 3, SEQ ID No. 5, SEQ ID No. 7 or one of their fragments, it is preferable that homologous, mainly specific, sequences have a percentage identity with one of the sequences of SEQ ID No. 1, SEQ ID No. 3, SEQ ID No. 5, SEQ ID No. 7 or one of their fragments equal to at least 80%, preferably 90% or 95%. These specific homologous sequences may contain, for example, sequences corresponding to the sequences of ORF1, ORF2, ORF3, PCV3. Likewise, these specific homologous sequences may correspond to changes associated with mutations within PCV3 strains, and in particular correspond to truncations, substitutions, deletions and/or additions of at least one nucleotide.

[95] The invention includes polypeptides encoded by a nucleotide sequence according to the invention, preferably a polypeptide the sequence of which is represented by a fragment, in particular a specific fragment; these six amino acid sequences correspond to polypeptides that can be encoded in accordance with one of three possible reading frames of the sequence SEQ ID No. 1 or the sequence SEQ ID No. 2.

[96] The invention also relates to polypeptides, characterized in that they contain a polypeptide selected from the amino acid sequences of SEQ ID No. 2, SEQ ID No. 4, SEQ ID No. 6, SEQ ID No. 8 or one of their fragments.

[97] The invention also relates to polypeptides, characterized in that they contain a polypeptide selected from: a) a specific fragment having at least 5 amino acids, a polypeptide of the amino acid sequence according to the invention; b) a polypeptide homologous to a polypeptide such as defined in a); c) a specific biologically active polypeptide fragment, such as defined in a) or b); and d) a polypeptide modified with a polypeptide such as those defined in a), b) or c).

[98] Also preferred among the polypeptides of the invention are those of the amino acid sequences SEQ ID No. 2, SEQ ID No. 4, SEQ ID No. 6 and SEQ ID No. 8, these polypeptides being particularly useful, in particular, for recognizing antibodies produced during PCV3 infection. Thus, these polypeptides have epitopes specific for PCV3 and can therefore be used, in particular, in the diagnostic field or as an immunogenic agent to provide protection to pigs against PCV3 infection.

[99] As used herein, the terms polypeptide, peptide and protein are used interchangeably.

[100] It should be understood that the invention does not relate to polypeptides in their natural state, i.e. they are not used in their natural environment, but may be isolated or purified from natural sources or produced by genetic recombination or, alternatively, chemical synthesis methods, and may therefore contain non-naturally occurring amino acids, as described below.

[101] By polypeptide fragment according to the invention is meant a polypeptide containing at least 5 consecutive amino acids, preferably 10 consecutive amino acids or 15 consecutive amino acids.

[102] As used herein, a specific polypeptide fragment is to be understood as a contiguous polypeptide fragment encoded by a specific fragment of the nucleotide sequence of the invention.

[103] Homologous polypeptide means polypeptides having certain modifications with respect to the natural polypeptide, such as, in particular, deletion, addition or substitution of at least one amino acid, truncation, extension, chimeric fusion and/or mutation. Among homologous polypeptides, preference is given to those whose amino acid sequence has at least 80%, preferably 90%, homology with the amino acid sequences of the polypeptides of the invention.

[104] By specific homologous polypeptide is meant homologous polypeptides, such as those defined above, having a specific polypeptide fragment of the invention.

[105] In the case of substitution, one or more consecutive or non-consecutive amino acids are replaced with "equivalent" amino acids. The expression "equivalent" amino acid means in the present description any amino acid capable of being replaced by one of the amino acids of the basic structure, without substantially changing the biological activity of the corresponding peptides and, thus, they can be defined as follows. These equivalent amino acids can be determined either by their structural homology to the amino acids they replace or by the results of possible comparative tests of biological activity between different polypeptides. As an example, we can mention the possibility of substitutions that can be made without significantly modifying the biological activity of the corresponding modified polypeptides, for example, substitutions of leucine with valine or isoleucine, aspartic acid with glutamic acid, glutamine with asparagine, arginine with lysine, etc., of course, that are also possible reverse substitutions under the same conditions.

[106] Specific homologous polypeptides also correspond to polypeptides encoded by specific homologous nucleotide sequences, such as those defined above, and thus include, as defined herein, polypeptides that are mutated or correspond to variants that may exist in PCV3 and that in particular correspond to truncations, substitutions, deletions and/or additions of at least one amino acid residue.

[107] A specific biologically active fragment of a polypeptide according to the invention, in particular, means a specific polypeptide fragment, as defined above, having at least one of the characteristics of the polypeptides according to the invention, in particular in the sense that it: is capable of inducing an immunogenic response, directed against PCV3; and/or capable of recognizing a specific antibody of a polypeptide of the invention; and/or is capable of binding to a polypeptide or nucleotide sequence of PCV3; and/or is capable of exhibiting physiological activity, even partial, such as, for example, spreading activity or structural (capsid) activity; and/or is capable of modulating, inducing or inhibiting the expression of the PCV3 gene or one of its variants and/or is capable of modulating the PCV3 replication cycle in the cell and/or organism of the host.

[108] The polypeptide fragments of the invention may correspond to isolated or purified fragments naturally present in PCV3, or corresponding fragments that can be obtained by cleavage of the polypeptide with a proteolytic enzyme such as trypsin or chymotrypsin or collagenase, or a chemical reagent such as cyanogen bromide (CNBr ), or alternatively, by placing said polypeptide in a very acidic environment, for example at pH 2.5. Such polypeptide fragments can also be easily obtained by chemical synthesis from hosts transformed with an expression vector of the invention containing a nucleic acid that provides expression of said fragments under the control of appropriate regulatory and/or expression elements.

[109] A "modified polypeptide" of a polypeptide of the invention means a polypeptide obtained by genetic recombination or chemical synthesis, as described below, having at least one modification with respect to the normal sequence. These modifications may particularly affect the parent amino acids of the polypeptide of the invention with respect to specificity, pathogenicity and/or virulence, or structural conformation and membrane insertion ability. Thus, it is possible to create polypeptides with equivalent, increased or decreased activity and equivalent, narrower or broader specificity. Among the modified polypeptides, mention must be made of polypeptides in which up to 5 amino acids may be modified, truncated at the N- or C-terminus, or even removed or added.

[110] As stated, the purpose of modifying a polypeptide is, in particular: to make it capable of modulating, inhibiting or inducing the expression of the PCV3 gene and/or capable of modulating the replication cycle of PCV3 in a cell and/or body of the host, which will allow its inclusion in vaccine compositions, and/or modify its bioavailability as a compound for therapeutic use.

[111] It has been shown that methods for achieving said modulation in eukaryotic or prokaryotic cells are well known to those skilled in the art. It should also be understood that nucleotide sequences encoding said modified polypeptides for said modulations can be used, for example, through the inventive vectors described below, for, for example, the prevention or treatment of pathologies associated with infection.

[112] The modified polypeptides described above can be obtained by combinatorial chemistry methods, which make it possible to sequentially change parts of the polypeptide before testing them on models, cell cultures or microorganisms, for example, to select the most active compounds or desired properties.

[113] Chemical synthesis also has the advantage of allowing the use of unnatural amino acids or non-peptide bonds. Thus, to improve the lifespan of the polypeptides of the invention, it may be of interest to use non-naturally occurring amino acids, such as the D-form, or amino acid analogues, particularly sulfur-containing forms.

[114] Finally, it is possible to integrate the structure of the polypeptides of the invention, their specific or modified homologous forms, into chemical structures of the polypeptide type or others. Thus, it may be of interest to obtain compounds at the N- and C-terminal ends that are not recognized by proteases.

[115] Nucleotide sequences encoding the polypeptide of the invention are also part of the invention.

[116] The invention also relates to nucleotide sequences that can be used as a primer or probe, characterized in that said sequences are selected from the nucleotide sequences of the invention.

[117] Cloning and sequencing of PCV3 allows the identification of nucleotide sequences by comparison with nucleotide sequences of other porcine circoviruses, which, unlike other sequences of fragments of these nucleic acids, are strictly specific to PCV3, and which correspond to the consensus sequence of porcine circoviruses other than PCV3. There is also a great need for nucleotide sequences used as a primer or probe that are specific for all other known and non-pathogenic porcine circoviruses.

[118] It should also be understood that the present invention also relates to specific polypeptides of known porcine circoviruses other than PCV3, encoded by the specified consensus nucleotide sequences, which can be obtained by purification from naturally occurring polypeptides by genetic recombination or by chemical synthesis using procedures well known to one skilled in the art and described in particular below. Thus labeled or unlabeled mono- or polyclonal antibodies to the specified specific polypeptides encoded by the specified consensus nucleotide sequences are also part of the invention.

[119] Said consensus nucleotide sequences, said corresponding polypeptides, and said antibodies to said polypeptides may be used in detection and/or identification procedures or kits, such as those described below, instead of or in addition to the nucleotide sequences, polypeptides or antibodies of the invention. specific for PCV3 type A and/or B.

[120] These protocols have been refined to differentiate between the cyclic monomeric forms of specific replicative forms of the virion or DNA during replication and the dimeric forms found in so-called tandemly arrayed molecular constructs.

[121] The invention also relates to the use of a nucleotide sequence according to the invention as a primer or probe for the detection and/or amplification of nucleic acid sequences.

[122] Thus, the nucleotide sequences of the invention can be used to amplify nucleotide sequences, in particular by PCR (polymerase chain reaction) (Erlich, 1989; Innis et al., 1990; RoIfs et al., 1991; White et al. , 1997). These oligodeoxyribonucleotide or oligoribonucleotide primers are advantageously at least 8 nucleotides in length, preferably at least 12 nucleotides, and more preferably at least 20 nucleotides in length. In addition to PCR, other alternative methods can be used to amplify the target nucleic acid.

[123] The nucleotide sequences of the invention, in particular the primers of the invention, can also be used in other procedures for amplifying a target nucleic acid, such as: the TAS (transcription-based amplification system) method described by Kwoh et al. in 1989; the 3SR (self-sustaining sequence replication) method described by Guatelli et al. in 1990; the NASBA (nucleic acid based amplification) method described by Kyivitis et al. in 1991; SDA (strand displacement amplification) method (Walker et al., 1992); TMA (transcription-mediated amplification) method.

[124] The polynucleotides of the invention can also be used in methods for amplifying or modifying a nucleic acid serving as a probe, such as: the LCR (ligase chain reaction) method described by Landegren et al. in 1988 and improved by Barany et al. in 1991, which uses a thermostable ligase; the RCR (chain reduction reaction) method described by Segev in 1992; the CPR (cyclic probe reaction) method described by Duck et al. in 1990; the Q-beta replicase amplification method described by Miele et al. in 1983 and improved, among others, by Chu et al. in 1986, Lizardi et al. in 1988, followed by Burg et al. and Stone et al. in 1996.

[125] If the target polynucleotide to be detected may be RNA, such as mRNA, a reverse-type enzyme may be used prior to an amplification reaction using at least one primer of the invention or prior to a detection procedure using at least one probe of the invention. transcriptase to produce cDNA from RNA contained in a biological sample. Thus, the resulting cDNA will serve as a target for the primer(s) or probe(s) used in the amplification or detection procedure in accordance with the invention.

[126] The detection probe can be selected to hybridize to the target sequence or an amplicon generated from the target sequence. With regard to the sequence, such a probe may advantageously have a sequence of at least 12 nucleotides, in particular at least 20 nucleotides and preferably at least 100 nucleotides.

[127] The invention also includes nucleotide sequences used as a probe or primer as described, characterized in that they are labeled with a radioactive compound or a non-radioactive compound. Unlabeled nucleotide sequences can be used directly as probes or primers, although, as a rule, the sequences are labeled with a radioactive element ( ³² P, ³⁵ S, ³ H, ¹²⁵ I) or a non-radioactive molecule (biotin, acetylaminofluorene, digoxigenin, 5-bromodeoxyuridine, fluorescein ) to produce probes that can be used in numerous applications. Examples of non-radioactive labeling of nucleotide sequences are described, for example, in French patent N 78.10975 or Urdea et al. or Sanchez-Pescador et al. in 1988. In the latter case, you can also use one of the tagging methods described in patents FR-2 422 956 and FR-2 518 755.

[128] The hybridization method can be carried out in various ways (Matthews et al., 1988). The most common method involves immobilizing a nucleic acid extract from cells onto a support (such as nitrocellulose, nylon, polystyrene) and incubating the immobilized target nucleic acid with a probe under well-defined conditions. After hybridization, excess probe is removed and the resulting hybrid molecules are determined by an appropriate method (by measuring radioactivity, fluorescence or enzymatic activity associated with the probe).

[129] The invention also includes the nucleotide sequences of the invention, characterized in that they are immobilized on a support through covalent or non-covalent bonds.

[130] According to another preferred method of using the nucleotide sequences of the invention, the latter can be immobilized on a support and thus serve to capture, by specific hybridization, a target nucleic acid obtained from a biological sample of interest. If necessary, the solid support is separated from the sample, and then the hybridization complex formed between said capture probe and the target nucleic acid is detected using a second probe, the so-called detection probe, labeled with an easily detectable element.

[131] Another aspect of the present invention is a vector for cloning and/or expression of a sequence, characterized in that it contains a nucleotide sequence according to the invention.

[132] In accordance with the invention, vectors that contain elements that provide expression and/or secretion of the specified nucleotide sequences in a particular host cell are also part of the invention. The vector must also contain a promoter, translation initiation and termination signals, and appropriate transcriptional regulatory regions. It must be stably maintained in the host cell and does not necessarily have specific signals that determine the secretion of the translated protein. These various elements are selected depending on the host cell used. To this end, the nucleotide sequences of the invention can be inserted into stand-alone replication vectors within a chosen host or integrated vectors of a chosen host. Such vectors can be produced by methods currently used by those skilled in the art, and the resulting clones can be introduced into a suitable host by standard methods such as, for example, lipofection, electroporation and heat shock. According to the invention, the vectors are, for example, plasmid or viral vectors. A preferred expression vector for the polypeptides of the invention is a baculovirus.

[133] These vectors can be used to transform host cells for cloning or expression of the nucleotide sequences of the invention. The invention also includes host cells transformed with a vector of the invention. These cells can be obtained by introducing into host cells a nucleotide sequence inserted into a vector such as defined above, then culturing said cells under conditions that allow replication and/or expression of the transfected nucleotide sequence. Host cells may be selected from prokaryotic or eukaryotic systems, such as, for example, bacterial cells (Olins and Lee, 1993), as well as yeast cells (Buckholz, 1993), animal cells, in particular mammalian cell cultures (Edwards and Aruffo, 1993), in particular Chinese hamster ovary (CHO) cells, as well as insect cells, in which procedures using, for example, baculoviruses can be used (Luckow, 1993). A preferred host cell for expression of the proteins of the invention is sf9 insect cells.

[134] The invention also relates to animals containing one of these transformed cells according to the invention. Transgenic animals of the invention overexpressing one or more PCV3 genes or portions of genes are preferably obtained from rats, mice or rabbits by methods well known to one skilled in the art, such as transfection of viruses or non-viruses. It is possible to obtain transgenic animals overexpressing one or more of these genes by transfecting multiple copies of these genes under the control of a strong universal promoter or a promoter that is selective for one tissue type. Similarly, transgenic animals can be produced by homologous recombination in embryonic cell strains, transferring these cell strains into embryos, selecting exposed chimeras at the reproductive line level, and growing said chimeras. Transformed cells, as well as transgenic animals according to the invention can be used in methods for producing recombinant polypeptides.

[135] It is now possible to produce recombinant polypeptides in relatively large quantities by genetic engineering using cells transformed with expression vectors of the invention or using transgenic animals of the invention. The present invention also includes methods for producing a polypeptide of the invention in recombinant form, characterized in that they use a vector and/or a cell transformed with a vector of the invention and/or a transgenic animal containing one of these transformed cells in accordance with the invention. Among these methods for producing the polypeptide of the invention in recombinant form, preferred are methods of production using a vector and/or a cell transformed by said vector and/or a transgenic animal containing one of said transformed cells containing the nucleotide sequence of the invention encoding the PCV3 polypeptide. Recombinant polypeptides prepared as above may also be present in either glycosylated or non-glycosylated form, and may or may not have a natural tertiary structure.

[136] A preferred option is to produce a recombinant polypeptide used for the carrier protein (chimeric protein). The advantage of this system is that it can stabilize and reduce proteolysis of the recombinant product, increase solubility during in vitro renaturation, and/or simplify purification if the fusion partner has an affinity for a particular ligand.

[137] More specifically, the invention relates to a method for producing a polypeptide of the invention, comprising the following steps: a) culturing the transformed cells under conditions allowing expression of a recombinant polypeptide of the nucleotide sequence of the invention; b) if necessary, recovering said recombinant polypeptide.

[138] When a transgenic animal according to the invention is used in a method for producing a polypeptide of the invention, the recombinant polypeptide is then recovered from said animal.

[139] The invention also relates to a polypeptide that can be obtained by the method described above.

[140] The invention also includes a method for producing a synthetic polypeptide, characterized in that it uses the amino acid sequence of the polypeptides of the invention. The invention also relates to a synthetic polypeptide obtained using the procedure in accordance with the invention.

[141] The polypeptides of the invention can also be prepared by methods that are conventional in the art of peptide synthesis. This synthesis can be carried out in a homogeneous solution or in the solid phase. For example, reference may be made to the homogeneous solution synthesis method described by Houben-Weyl in 1974. This method of synthesis consists of the sequential condensation, two by two, of successive amino acids in the required order, or the condensation of amino acids and fragments previously formed and already containing several amino acids in the appropriate order, or, alternatively, several fragments previously obtained in this way, it is understood that that it is necessary to ensure in advance the protection of all reactive functional groups carried by these amino acids or fragments, with the exception of the amine group in one and the carboxyl group in the other, or vice versa, which are usually involved in the formation of peptide bonds, especially after activation of the carboxyl group, in accordance with methods well known in the field of peptide synthesis. According to another preferred method of the invention, the method described by Merrifield can be used. To obtain a peptide chain in accordance with the Merrifield method, a polymer resin is used on which the first C-terminal amino acid of the chain is immobilized. This amino acid is immobilized on the resin through its carboxyl group, and the amino group is protected. Thus, the amino acids from which the peptide chain is formed are immobilized one after another, attached to the amino groups, from which each time the protection is first removed, of the peptide chain already formed and attached to the resin. Once the complete target peptide chain has been formed, the protecting groups of the various amino acids forming the peptide chain are removed and the peptide is separated from the resin using acid.

[142] The invention further relates to hybrid polypeptides having at least one polypeptide of the invention and a polypeptide sequence capable of inducing an immune response in humans or animals.

[143] Advantageously, the antigenic determinant is capable of inducing a humoral and/or cellular response. Such a determinant may contain the polypeptide of the invention in glycosylated form for use in the preparation of immunogenic compositions capable of inducing the synthesis of antibodies directed against multiple epitopes. Said polypeptides or glycosylated fragments thereof are also part of the invention. These hybrid molecules can be formed in part from a polypeptide carrier molecule or fragment thereof according to the invention, associated with a possible immunogenic part, in particular an epitope of diphtheria toxin, tetanus toxin, hepatitis B surface antigen (patent FR 79 21811), VP1 antigen of polio virus or any other viral or bacterial toxin or antigen. Procedures for the synthesis of hybrid molecules cover methods used in genetic engineering to construct hybrid nucleotide sequences encoding the desired polypeptide sequences. For example, a method for producing genes encoding fusion proteins described by Minton in 1984 can be cited. Said hybrid nucleotide sequences encoding a hybrid polypeptide, as well as hybrid polypeptides of the invention, characterized in that they are recombinant polypeptides obtained by expression of said hybrid nucleotide sequences, are also part of the invention.

[144] The invention also includes vectors characterized in that they contain one of these hybrid nucleotide sequences. Host cells transformed with said vectors, transgenic animals containing one of said transformed cells, as well as procedures for producing recombinant polypeptides using said vectors, said transformed cells and/or said transgenic animals are, of course, also part of the invention.

[145] The polypeptides of the invention, the antibodies of the invention described below, and the nucleotide sequences of the invention may advantageously be used in procedures for detecting and/or identifying PCV3 or porcine circovirus that is not PCV3 in a biological sample (biological tissue or fluid), in which it may be contained. These procedures, depending on the specificity of the polypeptides, antibodies and nucleotide sequences of the invention used, allow, in particular, the detection and/or identification of PCV3 or porcine circovirus that is not PCV3.

[146] The polypeptides of the invention can advantageously be used in a method for detecting and/or identifying PCV3 in a biological sample (biological tissue or fluid) in which it may be contained, characterized in that it includes the following steps: a) contacting this biological sample with the polypeptide or one of its fragments according to the invention (under conditions that provide an immunological reaction between the specified polypeptide and antibodies possibly present in the biological sample); and b) demonstrating the antigen-antibody complexes formed. Preferably, the biological sample is obtained from a fluid, such as serum, whole pig blood, or a biopsy. Any conventional procedure can be used to perform such detection of possible antigen-antibody complexes. By way of example, a preferred method includes enzyme immunoassays according to the ELISA method, immunofluorescence or radioimmunoassays (RIA) or equivalent.

[147] Thus, the invention also relates to the polypeptides of the invention labeled with a suitable label, such as an enzymatic, fluorescent or radioactive label. Such methods include, for example, the following steps: 1) precipitation of specified amounts of the polypeptide composition of the invention into the wells of a microtiter plate; 2) introduction into the indicated wells of serial dilutions of the serum or biological sample to be analyzed, different from that previously indicated; 3) microplate incubation; and 4) introducing into the wells of a microtiter plate labeled antibodies to porcine immunoglobulins, and the labeling of these antibodies is carried out using an enzyme selected from those that are capable of hydrolyzing the substrate by modifying the absorption of radiation by the latter, at least at a certain wavelength, for example, 550 nm, and detection of the amount of hydrolyzed support relative to the control test.

[148] The invention also relates to a kit or kit for the detection and/or identification of PCV3, characterized in that it contains the following elements: 1) a polypeptide according to the invention; 2) if necessary, reagents to create an environment favorable for an immunological or specific reaction; 3) if necessary, reagents capable of detecting antigen-antibody complexes produced by an immunological reaction between the polypeptide(s) of the invention and antibodies possibly present in the biological sample, these reagents may also be labeled or, in turn, be recognized by a labeled reagent, in particular in the case of an unlabeled polypeptide according to the invention; 4) if necessary, a biological control sample (negative control) devoid of antibodies recognized by the polypeptide of the invention; and 5) if necessary, a biological control sample (positive control) containing a specified amount of antibodies recognized by the polypeptide of the invention.

[149] The polypeptides of the invention enable the production of monoclonal or polyclonal antibodies which are characterized in that they specifically recognize the polypeptides of the invention. Advantageously, it is possible to obtain monoclonal antibodies from hybridomas according to the method described by Kohler and Milstein in 1975. Polyclonal antibodies can be produced, for example, by immunizing an animal, in particular a mouse, with the polypeptide or DNA of the invention associated with an immune response adjuvant, and then purifying the specific antibodies contained in the serum of the immunized animals on an affinity chromatography column with the previously immobilized polypeptide serving as as an antigen. The polyclonal antibodies of the invention can also be prepared by purifying the antibody contained in the serum of PCV3-infected pigs on an affinity chromatography column with a previously immobilized polypeptide.

[150] The invention also relates to mono- or polyclonal antibodies or fragments thereof or chimeric antibodies characterized in that they are capable of specifically recognizing the polypeptide of the invention. It is also possible for the antibodies described herein to be labeled in the same manner as previously described for the nucleic acid probes of the invention, for example, to have an enzymatic, fluorescent or radioactive label.

[151] The invention also relates to a method for detecting and/or identifying PCV3 in a biological sample, characterized in that it includes the following steps: a) contacting the biological sample (biological tissue or fluid) with a mono- or polyclonal antibody according to the invention (under conditions providing an immunological reaction between said antibodies and PCV3 polypeptides, including ORF1, ORF2 and/or ORF3, possibly present in the biological sample); and b) demonstrating the possible formation of an antigen-antibody complex.

[152] Also within the scope of the invention is a kit or kit for the detection and/or identification of PCV3, characterized in that it contains the following components: a) a polyclonal or monoclonal antibody according to the invention, if necessary labeled; b) if necessary, a reagent to create an environment favorable for the immunological reaction; c) if necessary, a reagent capable of detecting antigen-antibody complexes produced by an immunological reaction, which reagent is also capable of carrying a label or, in turn, being recognized by a labeled reagent, in particular if said monoclonal or polyclonal antibody is not labeled; and d) if necessary, reagents to perform cell lysis of the test sample.

[153] The present invention also relates to a method for detecting and/or identifying PCV3 in a biological sample, characterized in that it uses a nucleotide sequence according to the invention. More specifically, the invention relates to a method for the detection and/or identification of PCV3 in a biological sample, characterized in that it comprises the following steps: a) if necessary, extracting DNA from the biological sample for analysis; b) specific amplification of a DNA sample using at least one primer or pair of primers according to the invention; and c) demonstration of amplification products. The products can be detected, for example, by molecular hybridization using a nucleic acid probe according to the invention. This probe may advantageously be a labeled non-radioactive (cold probe) or radioactive element.

[154] For the purposes of the present invention, "DNA of a biological sample" or "DNA contained in a biological sample" should be understood either as DNA present in the biological sample in question, or possibly as cDNA obtained after the action of an enzyme such as reverse transcriptase on RNA, present in the specified biological sample.

[155] Another object of the present invention is the method of the invention, characterized in that it includes the following steps: a) contacting the nucleotide probe of the invention with a biological sample, DNA contained in the biological sample, which, if necessary, has been designed to provide hybridization under conditions allowing hybridization of the probe with the DNA of the specified sample; and b) demonstrating the hybrid formed between the nucleotide probe and the DNA of the biological sample.

[156] The present invention also relates to a method according to the invention, characterized in that it includes the following steps: a) contacting a nucleotide probe immobilized on a support in accordance with the invention with a biological sample, the DNA of the sample which, if necessary, has been engineered to provide hybridization under conditions allowing hybridization of the probe with the DNA of the specified sample; b) contacting the hybrid formed between the nucleotide probe immobilized on the support and the DNA contained in the biological sample, if necessary after excluding DNA from the biological sample that did not hybridize with the probe, with a nucleotide probe labeled in accordance with the invention; and c) demonstrating the new hybrid obtained in step b). According to a preferred embodiment of the previously defined detection and/or identification method, it is characterized in that, before step a), the DNA of the biological sample is first amplified using at least one primer according to the invention.

[157] The description also relates to a kit or kit for the detection and/or identification of PCV3, characterized in that it contains the following elements: a) a nucleotide probe according to the invention; b) if necessary, the reagents necessary to carry out the hybridization reaction; and c) if necessary, at least one primer according to the invention, as well as reagents necessary for the DNA amplification reaction.

[158] The invention also relates to a kit or kit for the detection and/or identification of PCV3 or porcine circovirus other than PCV3, characterized in that it contains the following components: a) a nucleotide probe, called a capture probe, according to the invention; b) an oligonucleotide probe, called a detection probe, according to the invention, and c) if necessary, at least one primer according to the invention, as well as reagents necessary for the DNA amplification reaction.

[159] The invention also relates to a kit or kit for the detection and/or identification of PCV3, characterized in that it contains the following elements: a) at least one primer according to the invention; b) if necessary, the reagents necessary to carry out the DNA amplification reaction; and c) if necessary, a component allowing the sequence of the amplified fragment to be verified, more particularly the oligonucleotide probe of the invention.

[160] The invention further relates to the use of a nucleotide sequence of the invention, a polypeptide of the invention, an antibody of the invention, a cell of the invention and/or an animal transformed in accordance with the invention to select an organic or inorganic compound capable of modulating, inducing or inhibiting gene expression and/or modify cellular replication of PCV3, or capable of inducing or inhibiting pathologies, including reducing the incidence and severity of clinical signs associated with PCV3 infection.

[161] The invention also includes a method for selecting compounds capable of binding to a polypeptide or one of its fragments of the invention, a nucleotide sequence of the invention, or capable of recognizing an antibody of the invention, and/or capable of modulating, inducing or inhibiting gene expression and/or modifying cellular replication PCV3, or capable of inducing or inhibiting pathology, including reducing the incidence and severity of clinical signs associated with PCV3 infection, characterized in that it includes the following stages: a) contacting the specified compound with the specified polypeptide, the specified nucleotide sequence or a cell transformed in accordance with with the invention and/or administering said compound to an animal transformed in accordance with the invention; and b) determining the ability of said compound to bind to said polypeptide or said nucleotide sequence or to modulate, induce or inhibit gene expression, or to modulate growth or replication of PCV3, or to induce or inhibit in said transformed animal pathologies associated with PCV3 infection (referred to as the activity of said connections). The compounds that may be selected are organic compounds such as polypeptides or carbohydrates, or any other known inorganic or inorganic compounds, or new organic compounds developed by molecular modeling methods and obtained by chemical or biochemical synthesis, these methods being known to one skilled in the art . These selected compounds can be used to modulate cellular replication of PCV3, and thereby combat infection caused by this virus; methods for determining these modulations are well known to those skilled in the art. This modulation may be accomplished, for example, by an agent capable of binding to a protein and thereby inhibiting or potentiating its biological activity, or capable of binding to a coat protein on the outer surface of said virus and blocking the entry of said virus into a host cell, or capable of stimulating the action of the immune system of an infected organism directed against the specified virus. This modulation may also be accomplished by an agent capable of binding to a DNA nucleotide sequence of said virus and blocking, for example, the expression of a polypeptide whose biological or structural activity is required for replication or proliferation of the virus in host cells of the host animal.

[162] The invention relates to compounds that can be selected by the selection method according to the invention.

[163] The invention also relates to a pharmaceutical composition containing a compound selected from the following compounds: a) a nucleotide sequence according to the invention; b) a polypeptide according to the invention; c) a vector, viral particle or cell transformed in accordance with the invention; d) antibodies according to the invention; and e) a compound that can be selected by the selection method of the invention; optionally in combination with a pharmaceutically acceptable carrier and, if necessary, one or more adjuvants of appropriate immunity.

[164] The invention also relates to an immunogenic and/or vaccine composition, characterized in that it includes a compound selected from the following compounds: a) a nucleotide sequence according to the invention; b) a polypeptide according to the invention; c) a vector or viral particle according to the invention; and d) cells according to the invention.

[165] In one embodiment, the vaccine composition of the invention is characterized in that it contains a mixture of at least two of the above compounds a), b), c) and d) and in that one of the two compounds is PCV3 .

[166] In another embodiment of the invention, the vaccine composition is characterized in that it contains at least one of the above compounds a), b), c) and d), which is PCV3. In another embodiment, the vaccine composition is characterized in that it contains at least one of the above compounds a), b), c) and d), which relates to PCV3 ORF2.

[167] A compound referred to as PCV3 should be understood to mean a compound derived from the genomic sequence of PCV3 and/or ORF1, ORF2 and ORF2 of PCV3.

[168] This disclosure further relates to an immunogenic and/or vaccine composition, characterized in that it contains at least one of the following compounds: 1) the nucleotide sequence of SEQ ID No.1, SEQ ID No.3, SEQ ID No.5 , SEQ ID No. 7 or one of their fragments or homologues; 2) a polypeptide with the sequence SEQ ID No.2, SEQ ID No.4, SEQ ID No.6, SEQ ID No.8 or one of their fragments, or modifications thereof; 3) a vector or viral particle containing the nucleotide sequence of SEQ ID No.1, SEQ ID No.3, SEQ ID No.5, SEQ ID No.7 or one of their fragments or homologues; 4) a transformed cell capable of expressing a polypeptide with the sequence SEQ ID No.2, SEQ ID No.4, SEQ ID No.6, SEQ ID No.8 or one of their fragments, or modifications thereof; or 5) mixtures of at least two of these compounds.

[169] This disclosure also includes an immunogenic and/or vaccine composition of the invention, characterized in that it contains said mixture of at least two of said compounds as a combination product for simultaneous, separate or chronic use for the prevention or treatment of PCV3 infection.

[170] In a preferred embodiment, the vaccine composition of the invention includes a mixture of the following compounds: 1) plasmid pcDNA3 containing a nucleic acid with the sequence SEQ ID No. 1; 2) plasmid pcDNA3 containing a nucleic acid with the sequence SEQ ID No.3, SEQ ID No.5 or SEQ ID No.7; 3) plasmid pcDNA3 containing nucleic acid encoding the GM-CSF protein; 4) recombinant baculovirus containing a nucleic acid with the sequence SEQ ID No.1; 5) a recombinant baculovirus containing a nucleic acid with the sequence of SEQ ID No.3, SEQ ID No.5 or SEQ ID No.7; and 6) if necessary, an appropriate immune adjuvant, in particular AIF™ adjuvant.

[171] This disclosure also relates to a pharmaceutical composition of the invention for the prevention or treatment of PCV3 infection.

[172] It is understood that "prophylaxis" as used herein includes complete prevention of PCV3 infection, but also includes reducing the severity or frequency of clinical signs associated with or caused by PCV3 infection. Such prophylaxis is also referred to herein as a protective action.

[173] This disclosure also relates to the use of the composition as described for the preparation of a medicament for the prevention or treatment of PCV3 infection.

[174] In another aspect, the invention relates to a vector, viral particle or cell of the invention for treating and/or preventing a disease using gene therapy.

[175] Finally, the invention includes the use of a vector, viral particle or cell of the invention to produce a medicament for the treatment and/or prevention of a disease using gene therapy.

[176] The polypeptides of the invention included in the immunogenic or vaccine compositions of the invention can be selected in ways known to one skilled in the art, such as, for example, depending on the ability of said translated polypeptides to stimulate T cells, for example, by their proliferation, or secretion of interleukins, and which lead to the production of antibodies directed against these polypeptides.

[177] In pigs, as in mice, which are administered a dose of the vaccine composition calculated on a weight basis comparable to the dose used in humans, the antibody response is tested by collecting serum and then testing by conventional methods for the formation of a complex between the antibody present in the serum and antigen of the vaccine composition.

[178] The pharmaceutical compositions of the invention may contain an effective amount of the compounds of the invention, i.e. a sufficient amount of the specified compound(s) to provide the desired effect, for example, modulation of PCV3 cellular replication. One skilled in the art will know how to determine such an amount depending on, for example, the age and weight of the person being treated, the severity of the pathology, possible side effects, and the use of a population-based effect test; these tests are known in these applications.

[179] According to the invention, said vaccine combinations are preferably combined with a pharmaceutically or veterinarily acceptable carrier and, if necessary, with one or more appropriate immune adjuvants.

[180] Currently, there are different types of vaccines to protect animals or humans from infectious diseases: attenuated live microorganisms (M. bovis - BCG for tuberculosis), inactivated microorganisms (influenza virus), cell extracts (Bordetella pertussis for whooping cough), recombinant proteins (surface antigen of hepatitis B virus), polysaccharides (pneumococcal). Vaccines derived from synthetic peptides or genetically modified microorganisms expressing heterologous antigens are under experimentation. More recently, recombinant plasmid DNAs carrying genes encoding protective antigens have been proposed as an alternative strategy for vaccine development. This type of vaccination is carried out by using a specific plasmid, derived from an E. coli plasmid, which does not replicate in vivo and which uniquely encodes the vaccine protein. Animals are immunized by simply injecting naked plasmid DNA into the muscle. This method results in in situ expression of the vaccine protein and cell type (CTL) and humoral type (antibody) immune responses. This dual induction of an immune response is one of the main advantages of the naked DNA vaccination method.

[181] The constitutive nucleotide sequence of the vaccine composition of the invention can be introduced into the host after attachment to compounds that promote the penetration of this polynucleotide into the cell or its transfer to the cell nucleus. The resulting conjugates can be encapsulated in polymer microparticles, as described in international application No. WO 94/27238 (Medisorb Technologies International).

[182] In another embodiment of the vaccine composition of the invention, the nucleotide sequence, preferably DNA, is complexed with DEAE-dextran (Pagano et al., 1967) or with nuclear proteins (Kaneda et al., 1989), lipids (Felgner et al. ., 1987) or encapsulated in liposomes (Fraley et al., 1980) or administered as a gel, which facilitates its transfection into cells (Midoux et al., 1993, Pastore et al., 1994). The polynucleotide or vector of the invention may also be suspended in a buffer solution or combined with liposomes.

[183] Such a vaccine is preferably prepared by the method described by Tacson et al. or Huygen et al. in 1996 or, alternatively, in the manner described by Davis et al. in international application No. WO 95/11307.

[184] Such a vaccine can also be obtained in the form of a composition containing the vector of the invention, which is under the control of regulatory elements that ensure its expression in humans or animals. For example, an in vivo polypeptide antigen expression vector, plasmid pcDNA3, or plasmid pcDNA1/neo, available from Invitrogen (R&D Systems, Abingdon, United Kingdom), can be used. You can also use the V1Jns.tPA plasmid described by Shiver et al. in 1995. Such a vaccine preferably includes, in addition to the recombinant vector, a physiological solution, for example a sodium chloride solution.

[185] As for vaccine formulations, they may contain adjuvants of appropriate immunity that are known to one skilled in the art, such as, for example, those described above.

[186] These compounds can be administered systemically, such as intravenously, intramuscularly, intradermally or subcutaneously, or orally. More preferably, the vaccine composition containing the polypeptides of the invention is administered intramuscularly, with food, or by repeated nebulization at specified intervals.

[187] Routes of administration, doses and optimal pharmaceutical forms can be determined in accordance with criteria that are usually taken into account when prescribing treatment adapted to the animal, such as, for example, age or weight, severity of the general condition, tolerance to treatment and observed secondary effects . Preferably, the vaccine of the present invention is administered in an amount that is protective or provides a protective effect against PCV3 infection.

[188] For example, in the case of a vaccine of the present invention containing a polypeptide encoded by the nucleotide sequence of the PCV3 genome, or a homolog or fragment thereof, the polypeptide is administered one or more times, which spreads over time, directly or through a transformed cell capable of expressing the polypeptide, in an amount of from 0.1 to 10 μg per kilogram of animal weight, preferably from about 0.2 to about 5 μg/kg, more preferably from about 0.5 to about 2 μg/kg per dose.

[189] The present invention also relates to the use of the PCV3 nucleotide sequences of the invention for the generation of autoreplicative retroviral vectors and their therapeutic use, particularly in the field of in vivo gene therapy.

[190] There is no need to further demonstrate the feasibility of gene therapy in humans, and this also applies to numerous therapeutic applications such as genetic diseases, infectious diseases and cancers. Numerous prior art documents describe methods of using gene therapy, in particular using viral vectors. In general, vectors are produced by deleting at least some of the viral genes, which are replaced by genes of therapeutic interest. Such vectors can be propagated in a complementing cell line that provides remote viral functions in trans, allowing the generation of a replication-defective viral vector particle but capable of infecting a host cell. Today, retroviral vectors are the most widely used, and their mode of infection is well described in the literature available to one skilled in the art.

[191] The principle of gene therapy is to deliver a functional gene, called the gene of interest, whose RNA or corresponding protein will produce the desired biochemical effect in the target cells or tissues. On the one hand, gene injection allows for the long-term expression of complex and unstable molecules, such as RNA or proteins, which are extremely difficult or even impossible to obtain or directly introduce. On the other hand, controlled insertion of the desired gene inside target specific cells makes it possible to regulate the expression product in certain tissues. To do this, it is necessary to be able to insert the desired therapeutic gene inside selected cells and, thus, have an available route of administration that can specifically target the selected cells or tissues. Some preferred genes of interest from the point of view of the present invention are those encoding ORF1, ORF2 or ORF3.

[192] Among gene introduction methods, gene introduction methods are widely used, such as, for example, microinjection, especially naked plasmid DNA injection, electroporation, homologous recombination, the use of viral particles such as retroviruses. However, when used in vivo , recombinant retroviral gene transfer systems at the same time have weak infectious ability (insufficient concentration of viral particles) and lack of specificity for the selected target cells.

[193] The production of viral vectors specific to certain cell types, having tissue-specific tropism and containing a gene of interest that can be adequately translated in the target cell, is realized, for example, by fusion of a specific ligand of the target host cells with the N-terminal part of the surface envelope protein PCV3. Mention may be made, for example, of constructing retroviral particles having a CD4 molecule on the surface of the envelope to target human cells infected with the HIV virus, viral particles having a peptide hormone fused to the envelope protein to specifically infect cells expressing the corresponding receptor, or alternatively , viral particles having a fusion polypeptide capable of attaching to the epidermal growth factor (EGF) receptor. In another approach, single-chain antibody fragments to surface antigens of target cells are introduced by fusion with the N-terminal part of the envelope protein.

[194] For purposes of the present invention, a gene of interest for use in the invention may be obtained from a eukaryotic or prokaryotic organism or from a virus by any conventional method. Preferably, it is capable of producing an expression product having a therapeutic effect, and may be a product homologous or, alternatively, heterologous to the host cell. Within the scope of the present invention, the gene of interest may encode (1) an intracellular or (2) membrane product present on the surface of the host cell or (3) secreted outside the host cell. Therefore, it may contain appropriate additional elements, such as, for example, a sequence encoding a secretion signal. These signals are known to one skilled in the art.

[195] In accordance with the objectives of the present invention, the gene of interest may encode a protein corresponding to all or part of a native protein found in nature. It may also be a chimeric protein, for example resulting from the fusion of polypeptides of different origins, or a mutant having improved and/or modified biological properties. Such a mutant can be obtained by conventional biological methods by substitution, deletion and/or addition of one or more amino acid residues.

[196] Thus, the invention relates to vectors characterized in that they contain the PCV3 nucleotide sequence of the invention and in that they additionally contain a gene of interest.

[197] Likewise, the present invention relates to viral particles obtained from the specified vector in accordance with the invention. It also relates to methods for producing viral particles in accordance with the invention, characterized in that they use a vector according to the invention, including viral pseudoparticles (VLP, virus-like particles).

[198] The invention also relates to animal cells transfected with a vector of the invention. The invention also provides animal cells, in particular mammalian cells, infected with a viral particle according to the invention.

[199] One preferred vaccine uses a DNA clone of a live chimeric virus, specifically a clone containing immunogenic PCV3 genes cloned into a non-pathogenic PCV1 backbone. The predominantly live chimeric virus, which is naturally avirulent when created by genetic engineering, does not require lengthy attenuation procedures. The virus is used exclusively as a live but non-pathogenic replicating virus that produces immunogenic anti-PCV3 proteins during replication, which can then elicit a full range of immune responses against pathogenic PCV3.

[200] As an additional advantage, the live chimeric virus of the present invention provides a genetically stable vaccine that is easier to manufacture, store and deliver compared to other types of attenuated vaccines. Avirulent or attenuated chimeric virus vaccines are generally considered as safe, if not more so, than traditionally modified live vaccines. For example, the ChimeriVax-JE vaccine against Japanese encephalitis virus (JEV), which is a genetically engineered derivative of the YFV17D yellow fever virus vaccine, has been shown to replace the genes encoding the structural prM and E proteins of YFV17D with the corresponding genes of an attenuated strain SA14-14-2 JEV is genetically stable after prolonged passage both in vitro and in vivo . Another chimeric virus vaccine, ChimeriVax-D2, against dengue virus type 2, which is an attenuated chimeric yellow fever (YF) virus type 2 (dengue-2 virus), has been found to be genetically stable; there is also a report that its sequence did not change after 18 passages in Vero cells.

[201] Another preferred vaccine of the present invention uses suitable plasmids to deliver a non-pathogenic chimeric DNA clone to pigs. Unlike a traditional vaccine, which uses a live or killed cell culture in which the whole virus has been propagated, the present invention involves direct inoculation of pigs with plasmid DNA containing an infectious chimeric viral genome.

[202] Additional genetically engineered vaccines used in the present invention are prepared by methods known in the art. Such methods include, but are not limited to, additional methods of processing recombinant DNA, modifying or substituting amino acid sequences of recombinant proteins, and the like. Genetically engineered vaccines based on recombinant DNA technology are produced, for example, by identifying alternative parts of the viral gene that encode proteins responsible for inducing a stronger immune or protective response in pigs (eg, proteins derived from ORF1, ORF2, ORF3). Such identified genes or immunodominant fragments can be cloned into standard protein expression vectors, such as a baculovirus vector, and can be used to infect appropriate host cells. The host cells are cultured, thereby allowing expression of the desired vaccine proteins, which can be purified to the desired extent and incorporated into a suitable vaccine product.

[203] If clones retain the undesirable natural ability to cause disease, the nucleotide sequences in the viral genome responsible for any residual virulence can be precisely determined and an avirulent virus can be genetically engineered using, for example, site-directed mutagenesis. Site-directed mutagenesis allows one or more nucleotides to be added, deleted, or changed. An oligonucleotide containing the required mutation is synthesized and annealed to a portion of single-stranded viral DNA. The hybrid molecule resulting from this procedure is used to transform bacteria. The double-stranded DNA containing the corresponding mutation is then isolated and used to obtain full-length DNA by ligating the latter's restriction fragments, which is then transfected into a suitable cell culture. Ligation of the genome into a suitable transfer vector can be accomplished by any standard method known to those skilled in the art. Transfection of the vector into host cells to produce viral progeny can be carried out by any conventional method, such as calcium phosphate or DEAE-dextran transfection, electroporation, protoplast fusion, and other well known methods. The cloned virus then exhibits the desired mutation. Alternatively, two oligonucleotides can be synthesized that contain the appropriate mutation. They can be annealed to form double-stranded DNA, which can be inserted into viral DNA to produce full-length DNA.

[204] Genetically engineered proteins used in vaccines, for example, can be expressed in insect cells, yeast cells or mammalian cells. Genetically engineered proteins, which can be purified or isolated by conventional methods, can be directly administered to pigs to provide protection against viral infection or post-weaning multisystem wasting syndrome (PMWS) caused by PCV3. An insect cell line (eg, HI-FIVE) can be transformed with a transfer vector containing nucleic acid molecules derived from the virus or copied from the viral genome that encodes one or more immunodominant proteins of the virus. The transfer vector includes, for example, linearized baculovirus DNA and a plasmid containing the desired polynucleotides. A host cell line can be cotransfected with linearized baculovirus DNA and plasmid to produce recombinant baculovirus.

[205] Alternatively, DNA from a pig suffering from pneumonia, reproductive failure, PDNS and/or PMWS that encodes one or more capsid proteins, PCV3, can be inserted into live vectors such as a poxvirus or adenovirus and used as a vaccine. -infectious molecular DNA clone or PCV-cloned chimeric DNA genome.

[206] An immunologically effective amount of the vaccines or immunogenic compositions of the present invention is administered to a pig in need of protection against viral infection, pneumonia, reproductive failure, PDNS and/or PMWS. The immunologically effective amount or immunogenic amount administered to a pig can be easily determined or easily titrated by routine testing. An effective amount is an amount that produces a sufficient immunological response to the vaccine to protect a pig exposed to the virus that causes PMWS. Preferably, the pig is protected to the extent that all adverse physiological symptoms or effects of the viral disease are reduced or alleviated to a significant extent or completely prevented.

[207] The vaccine can be administered as a single dose or in repeated doses, with single doses being preferred. Single-dose vaccines provide protection after a single dose without the need for any booster or follow-up doses. Protection may include completely preventing the onset of clinical signs of infection or reducing the severity, duration or likelihood of one or more clinical signs of infection. Dosages may vary, for example, from about 1 microgram to about 1000 micrograms of plasmid DNA containing the infectious chimeric DNA genome (depending on the concentration of the immunoactive vaccine component), preferably from 100 to 200 micrograms of the chimeric DNA clone PCV1-3, but should not contain an amount of virus-based antigen sufficient to cause an adverse reaction or physiological symptoms of a viral infection. Methods are known in the art to determine or titrate appropriate doses of active antigenic agent to determine minimum effective doses based on pig weight, antigen concentration, and other typical factors. It is preferable to use an infectious chimeric viral DNA clone as the vaccine, or it is possible to produce a live infectious chimeric virus in vitro and then use the live chimeric virus as a vaccine. In this case, the pig can be administered, for example, from 50 to 10,000 50% tissue culture infectious dose (TCID ₅₀ ) of the live chimeric virus.

[208] It is advisable to administer the vaccine to a pig that has not yet been infected with PCV. A vaccine containing a chimeric DNA clone of PCV1-3 or its other antigenic forms can be conveniently administered intranasally, transdermally (i.e. applied to the surface of the skin for systemic absorption), parenterally, etc. The parenteral route of administration includes, but is not limited to, intramuscular, intravenous, intraperitoneal, intradermal (i.e., injected or otherwise applied under the skin) administration, and the like. Because intramuscular and intradermal inoculations have been successful in other studies using viral infectious DNA clones, these routes of administration are most preferred, in addition to the convenience of intranasal administration. Although less convenient, it is also possible to administer the vaccine to the pig by lymph node inoculation. One unique, preferred route of administration involves directly injecting a pig with plasmid DNA containing the PCV1-3 chimera into muscle, subcutaneously, lymph nodes, etc.

[209] When administered as a liquid, the present vaccine may be prepared as an aqueous solution, syrup, elixir, tincture, or the like. Such preparations are known in the art and are typically prepared by dissolving the antigen and other typical additives in appropriate carrier or solvent systems. Suitable carriers or solvents include, but are not limited to, water, saline, ethanol, ethylene glycol, glycerin, etc. Typical additives are, for example, certified colors, flavors, sweeteners and antimicrobial preservatives such as thimerosal (sodium ethyl mercury thiosalicylate). Such solutions can be stabilized, for example, by the addition of partially hydrolyzed gelatin, sorbitol or cell culture medium and can be buffered by conventional methods using reagents known in the art, such as sodium hydrogen phosphate, sodium dihydrogen phosphate, potassium hydrogen phosphate, potassium dihydrogen phosphate, mixtures thereof and so on.

[210] Liquid formulations may also include suspensions and emulsions that contain suspending or emulsifying agents in combination with other standard components of the drug formulation. These types of liquid compositions can be prepared by conventional methods. Suspensions, for example, can be prepared using a colloid mill. Emulsions, for example, can be prepared using a homogenizer.

[211] Parenteral products intended for injection into body fluids require proper isotonicity and pH buffering to appropriate pH levels of body fluids in pigs. Isotonicity can be adjusted accordingly with sodium chloride and other salts as needed. Suitable solvents, such as ethanol or propylene glycol, can be used to increase the solubility of the ingredients in the formulation and the stability of the liquid formulation. Additional additives that may be used in the present vaccine include, but are not limited to, dextrose, conventional antioxidants, and conventional chelating agents such as ethylenediaminetetraacetic acid (EDTA). Parenteral dosage forms also require sterilization before use.

[212] In another embodiment, the present invention relates to a protein encoded by a chimeric DNA molecule. This protein can be administered alone or in addition to other compositions described herein. In preferred forms, the protein is administered in an amount of from about 0.2 to about 400 μg/ml. The chimeric DNA molecule disclosed herein is preferred.

[213] A specific PCV3 DNA clone is designed according to the invention to allow the production of a biologically pure and homogeneous virus stock solution for studying pathogenesis and developing non-pathogenic chimeric vaccines. The use of this molecular DNA clone and the biologically pure and homogeneous PCV3 virus stock solution derived from the molecular DNA clone allows the dynamics of clinical disease, viral spread and pathological lesions associated with PCV3 infection to be characterized more accurately than before, providing the opportunity for development the desired vaccine products of the present invention.

[214] A molecular clone of PCV3 is generated by ligating two copies of the complete PCV3 genome in tandem into the pSK vector. In sharp contrast to the single-copy genome known in the art, the PCV3 infectious DNA clone obtained by the methods described herein contains two complete copies of the PCV3 genome ligated together in a tandem repeat. Ligation of two copies of the genome in tandem produces a similar circular genome that mimics the conventional circular genome of PCV3. The advantage of having two copies of the genome in tandem in a PCV3-infectious DNA clone is increased replication during in vitro and in vivo infection of the DNA clone. Thus, the clone of the invention works more efficiently, more efficiently, than the previous single-copy genome.

[215] Infecting animals with a molecular viral clone is extremely useful for studying the genetic determinants of viral replication and virulence in the host. PCV3 has been identified as the causative agent of pneumonia, reproductive failure, PMWS and PDNS. PMWS and PDNS are complex disease syndromes in pigs, and several factors may be involved in the clinical manifestation of PMWS and/or PDNS. However, the difficulty of obtaining a biologically pure form of PCV3 due to the presence of other commonly occurring porcine agents in tissue homogenates of affected pigs has prevented definitive characterization of clinical diseases and pathological lesions that were exclusively associated with PCV3 infection. This will be the first time that a PCV3-infectious molecular DNA clone will be constructed and used to characterize the disease and pathological lesions associated with PCV3 infection by direct in vivo transfection of the molecular clone into pigs.

[216] A homogeneous stock solution of live PCV3 virus derived from a molecular clone has been shown to be infectious in vitro when transfected into PK-15 cells. Cloned PCV3 genomic DNA is also infectious when directly injected into the liver and superficial iliac lymph nodes of pathogen-free (SPF) pigs. Animals injected with cloned PCV3 plasmid DNA typically develop infection and disease similar to those resulting from intranasal inoculation of a homogeneous infectious stock solution of live PCV3 virus. Seroconversion to PCV3-specific antibody was detected in the majority of pigs from the inoculated groups at 35 days post-inoculation (DPI).

[217] The onset and duration of viremia in pigs inoculated with the chimeric PCV1-3 DNA clone is the same as in pigs inoculated with the non-pathogenic PCV1 DNA clone, whereas viremia in pigs inoculated with the PCV3 clone appears earlier and is longer lasting. From day 14 DPI onwards for approximately 2-6 weeks, viremia is observed in most animals infected with PCV3. Likewise, the majority of inoculated pigs, when necropsied at 35 days DPI, were seroconverted to PCV3 antibodies. PCV3 antigen is found in various tissues and organs of inoculated pigs. Large lesions are limited to the lungs and lymph nodes and are characterized by systemically enlarged yellowish-brown lymph nodes, lungs that do not collapse, and soft, multifocal, tan-colored areas of consolidation. Large lymph node lesions in both pigs infected with non-pathogenic PCV1 and pigs infected with chimeric PCV1-3 are mild and observed in only a few animals, whereas in pigs infected with pathogenic PCV3 there are cases of moderate to severe swelling and discoloration lymphoid tissues are more common. Statistical analysis shows that the number of extensive lesions in the lymph nodes of animals inoculated with chimeric PCV1-3 is the same as in pigs inoculated with non-pathogenic PCV1. At 21 DPI, pigs inoculated with PCV3 exhibit severe lesions that are statistically more severe than those inoculated with PCV1 and chimeric PCV1-3. Histopathological lesions and PCV3-specific antigen are found in numerous tissues and organs, including the brain, lungs, heart, kidneys, tonsils, lymph nodes, spleen, ileal gland and liver of infected (infected) pigs. In the case of the molecular DNA clone PCV3, as well as the infectious virus obtained in vitro from the molecular DNA clone, histopathological lesions in many tissues and organs are similar to those observed in PMWS. When examined microscopically at both 21 and 49 DPI, animals infected with chimeric PCV1-3 had statistically less significant microscopic damage than animals infected with PCV3. The rates of microscopic lesions in the lymph nodes of pigs inoculated with chimeric PCV1-3 are similar to those of animals inoculated with non-pathogenic PCV1, reciprocal chimeric PCV3-1, and uninfected control animals. Moderate to severe microscopic lesions are observed in many tissues of animals infected with pathogenic PCV3, including lungs, liver, lymphoid tissue, spleen, brain, heart, kidneys, and tonsils. However, in animals inoculated with chimeric PCV1-3, mild to moderate microscopic lesions are limited to liver, lymph node, and kidney tissues only.

[218] The availability of the infectious PCV3 DNA clone described herein allows the development of a genetically engineered attenuated vaccine to prevent PCV3 and PDNS and PMWS infection in pigs.

[219] The structural and functional relationships of the PCV genes can be better understood through the presence of infectious DNA clones PCV3, PCV1, chimeric PCV1-3 and reciprocal chimeric PCV3-1 described herein.

[220] Construction of a PCV3 infectious molecular DNA clone and demonstration of infection by direct injection of cloned PCV3 plasmid DNA into the liver and lymph nodes of pigs in the context of the present invention will be useful for the study of PCV3. This in vivo transfection system allows for improved study of the structural and functional relationship of PCV3 genes by using recombinant plasmids constructed in vitro to test different regions or genes of PCV3 for their role in viral replication and pathogenesis in the host. PCV3 replication and pathogenesis can be studied in vivo without the need to generate infectious virus stocks by propagating PCV3 in cell cultures. This is advantageous because viral variants can be targeted for serial passages in cell cultures. Another advantage of using cloned PCV3 genomic DNA instead of live virus in animal studies is the relative ease of quantifying the inoculated dose. The amount of cloned PCV3 DNA used to inoculate animals can be easily determined using a spectrophotometer, whereas the dose of live PCV3 virus requires titration of infectivity in cell cultures and confirmation of infection by IFA. Direct injection of cloned PCV3 plasmid DNA into animals eliminates the problems associated with the presence of other topical agents in the pig when introducing tissue homogenate for animal studies.

[221] In one aspect of the present invention, the immunogenic capsid protein gene ORF2 is switched between pathogenic PCV3 and non-pathogenic PCV1 to produce a unique structure of a chimeric viral DNA clone PCV1-3. Surprisingly, the chimeric PCV1-3 infectious clone replicates, expresses the immunogenic capsid protein antigen ORF2 in vitro and in vivo , and induces a specific antibody response against PCV3 ORF2 while maintaining the nonpathogenic nature of PCV1. The chimeric PCV1-3 infectious DNA clone has the ability to induce a strong immune response against PCV3 while causing only limited infection with mild pathological lesions similar to those of non-pathogenic PCV1. In vaccine development, the relative ease of storage and stability of cloned DNA, as well as the economies of scale production of recombinant PCV3 plasmid DNA and chimeric PCV1-3 DNA clone, provide an attractive means of delivering live, infectious viral DNA vaccine or genetically modified attenuated viral vaccines to pigs. . Therefore, the chimeric PCV1-3 infectious DNA clone described herein is a useful vaccine candidate against PCV3 infection and PMWS.

[222] It should be understood that all scientific and technological terms used herein have the same meaning as they are commonly used by those skilled in the art. For the purposes of the present invention, the term "infectious" means that the virus replicates in pigs, regardless of whether it causes any disease. "SPF" refers to pathogen-free pigs.

[223] "Gnotobiotic" pigs refer to pigs that are free of germs. The terms "PCV3 plasmid DNA", "PCV3 genomic DNA" and "PCV3 molecular DNA" are used interchangeably to refer to the same cloned nucleotide sequence.

[224] The following examples demonstrate certain aspects of the present invention. However, it should be understood that these examples are provided for purposes of illustration only and are not intended to be complete with respect to the terms and scope of the present invention. It should be noted that when typical reaction conditions are specified (eg, temperature, reaction time, etc.), conditions both above and below the specified ranges can also be used, although they are usually less convenient. The Examples were performed at room temperature (about 23°C to about 28°C) and at atmospheric pressure. All parts and percentages mentioned herein are by weight and all temperatures are expressed in degrees Celsius unless otherwise noted. In addition, unless otherwise indicated, all components of the invention are to be understood within the scope of the terms "comprising", "consisting essentially of" and "consisting of" used in the claims in the sense in which such terms are generally used in patent applications. documents

[225] Another aspect of the present invention is the production of combination vaccine(s) or immunogenic compositions. Such combinations can be made from various vaccine components described in this application. For example, the vaccine of the present invention may include both protein portions and DNA portions of PCV3, as described herein, administered simultaneously or separately. In addition, combinations can be made from the PCV3 vaccine components described herein and antigens from other pathogens such as those described above.

[226] In another aspect, the vaccine or immunogenic composition is first dehydrated. If the composition is first lyophilized or otherwise dehydrated, the composition is rehydrated in an aqueous solution (for example, saline, PBS) or non-aqueous solutions (for example, an oil emulsion (single or double emulsion based on mineral oil or vegetable/metabolizable oil), aluminum-based or carbomer-based adjuvant).

[227] According to the present invention, an effective amount of a combination vaccine administered to a pig provides effective immunity or protection against microbiological infections caused by PCV3 and at least one additional pathogen. Preferred combinations of antigens for the treatment and prevention of microbiological diseases in pigs are listed above.

[228] In another embodiment, the combination vaccine is administered to pigs in one or two doses 2 to 4 weeks apart. For example, the first administration is performed on an animal between about 2-3 weeks of age and about 8 weeks of age. The second administration is performed approximately 1-4 weeks after the first administration of the first vaccination. In another embodiment, the booster vaccination is performed between 3 and 12 months after the second dose. Subsequent doses of the vaccine are preferably administered every 6 months. In another preferred embodiment, animals vaccinated before reaching 2-3 weeks of age should be revaccinated. Subsequent doses of the vaccine are preferably given annually. In the event that one component of a combination vaccine is effective after a single dose, that component only needs to be administered once with the other component(s) administered according to their preferred schedule.

[229] The amount of effective combination vaccine depends on the vaccine ingredients and the mode of administration. When an inactivated virus or modified live virus preparation is used in a combination vaccine, the amount in the vaccine is from about 10 ² to about 10 ⁹ TCID ₅₀ per dose, preferably from about 10 ³ to about 10 ⁸ TCID ₅₀ per dose, more preferably from about 10 ⁴ to approximately 10 ⁸ TCID ₅₀ per dose. In general, inactivated antigen is usually used in larger quantities than live modified viruses. Typically, when a bacterial antigen is used in a combination vaccine, the vaccine contains from about 10 ³ to about 10 ⁹ colony forming units (CFU) per dose, preferably from about 10 ⁴ to about 10 ⁸ (CFU) per dose, more preferably from about 10 ⁵ to approximately 10 ⁶ (CFU) per dose. Typically, subunit vaccines are administered at an antigen incorporation level of at least 0.2 μg of antigen per dose, preferably from about 0.2 to 400 μg/dose, more preferably from 0.3 to 200 μg/dose, even more preferably from about 0. 35 to 100 mcg/dose, even more preferably from about 0.4 to about 50 mcg/dose, even more preferably from about 0.45 to about 30 mcg/dose, even more preferably from about 0.6 to about 15 mcg/dose dosage, even more preferably from about 0.75 to about 8 μg/dose, even more preferably from about 1.0 to about 6 μg/dose, and even more preferably from about 1.3 to about 3.0 μg/dose. For example, the antigen inclusion level for PCV3 ORF2 antigen, preferably PCV3 ORF2 protein, as provided herein, contains from about 2 to about 150 μg, preferably from about 2 to about 60 μg, even more preferably from about 2 μg to about 50 μg , even more preferably from about 2 μg to about 40 μg, even more preferably from about 2 μg to about 30 μg, even more preferably from about 2 μg to about 25 μg, even more preferably from about 2 μg to about 20 μg, yet more preferably from about 4 μg to about 20 μg and even more preferably from about 4 μg to about 16 μg.

[230] The composition of the invention can be administered intradermally, intratracheally or intravaginally. The composition is preferably applied intramuscularly or intranasally. It may be more preferable to administer the pharmaceutical composition described above into the animal by intravenous injection or by direct injection into target tissues. For systemic use, intravenous, intravascular, intramuscular, intranasal, intraarterial, intraperitoneal, oral or intrathecal routes of administration are preferred. More local application is possible by subcutaneous, intradermal, transdermal, intracardiac, intracavitary, intragastric, intramuscular administration or injection directly into or near the tissue to be treated (connective, bone, muscle, nervous, epithelial tissue). Depending on the desired duration and effectiveness of treatment, the compositions of the invention can be administered one or more times, as well as periodically, for example daily for several days, weeks or months and in different doses.

Example 1

[231] In this example, a qPCR assay is developed to specifically detect the PCV3 capsid protein gene.

[232] Viral DNA was isolated from clinical samples using the MagMax-96 Complete Nucleic Acid Isolation Kit according to the manufacturer's protocol. Nucleic acid was extracted from formalin-fixed, paraffin-embedded tissues using the QIAamp DNA FFPE Tissue kit according to the manufacturer's instructions (Qiagen, Valencia, CA). A 5′ nuclease assay was performed to target the 112 bp region. PCV3 gene nucleic acid in samples: probe, 5'-FAM-ACC CCA TGG-Zen-CTC AAC ACA TAT GAC C-Iowa Black-3' (SEQ ID No.14); forward primer, 5'-AGT GCT CCC CAT TGA ACG-3' (SEQ ID No. 13); reverse primer, 5'-ACA CAG CCG TTA CTT CAC-3' (SEQ ID No. 12). Quantitative PCR was performed using the Qiagen Quantitect PCR kit under the following conditions: 95°C, 15 minutes; and 45 cycles at 94°C for 15 seconds and 60°C for 60 seconds. The sensitivity and specificity of the assay were determined by serial dilution of a plasmid (pSF-CMV-cap) containing the complete PCV3 cap gene cloned into pSF-CMV-AMP (Oxford Genetics, UK) and PCV2 nucleic acid extracted from cell culture. Cycle threshold (Ct) values determined by serial dilution of the plasmid were used to generate a standard curve for calculations to determine genomic copies/ml (gc/ml).

[233] The complete genome for PCV3 was determined from a pool of homogenates of germ tissue samples obtained from a farm with a PDNS outbreak and from a sample in a prevalence study using Sanger sequencing of four overlapping amplicons obtained using the primers shown in Table 1. PCR was performed using the TaKaRa Taq™ kit as follows: 94°C, 4 minutes; then 40 cycles of 94°C, 20 seconds; 50°C, 30 seconds; 72°C, 1 minute; and 72°C for 5 minutes. Sequencing to confirm selection of PCV3 positive samples was performed using the 330 bp internal gene primer set: 5'-CCA CAG AAG GCG CTA TGT C-3' (SEQ ID No.16) and 5'-CCG CAT AAG GGT CGT CTT G-3' (SEQ ID No.17). PCR reactions of the cap gene were performed using the TaKaRa Taq™ kit as described above. PCR products were Sanger sequenced for verification.

Example 2

[234] In this example, a virus was isolated.

[235] The virus was isolated from testicular cells (ST) and renal cells (PK-15) of pigs maintained in minimal essential medium (MEM) supplemented with L-glutamine and 5% fetal serum. Cells were seeded into 6-well plates (60-80% confluent), and 100 μl of sample was inoculated into 1 ml of viral replacement medium, which consisted of MEM and penicillin-streptomycin solution. Cells were observed daily for cytopathic effects, and PCV3 growth was monitored by qPCR and immunofluorescence.

Example 3

[236] This example demonstrates the cloning, expression and purification of the PCV3 capsid protein.

[237] To create a recombinant PCV3 cap construct, primers were designed to amplify the portion of the PCV3 gene encoding amino acids (aa) 35-214: F, 5'-AAA AAA GCT AGC GCT GGA ACA TAC TAC ACA-3' (SEQ ID No. 18); R, 5'-AAA AAA GAA TTC TTA GAG AAC GGA CTTGTA ACG-3' (SEQ ID No. 19). The 5' ends of the forward and reverse primers contained NheI and EcoRI restriction sites (underlined), respectively. PCR products were cloned into the pET28a vector (Novagen, Madison, WI) to allow expression of the N-terminal truncated cap PCV3 as an N-terminal 6x histidine (His) recombinant protein in E. coli . Plasmid pET28a-cap was transformed into BL21(DE3) E. coli cells and expressed as previously described. Following expression, bacteria were collected by centrifugation and lysed using B-PER reagent (Pierce, Rockford, IL) after purification in Ni-NTA agarose (Qiagen, Valencia, CA) according to the manufacturer's instructions. The purity and identity of the recombinant protein were assessed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) under denaturing conditions, as well as by Western blotting to detect a His tag at the N-terminus of the recombinant protein.

Example 4

[238] This example demonstrates the preparation and in vitro characterization of a monoclonal antibody to the PCV3 capsid protein.

[239] BALB/c mice were immunized with purified, truncated capsid protein (35-214aa). Mice were inoculated with 50 μg of antigen mixed with Freund's incomplete adjuvant biweekly for eight weeks. The mouse splenocyte cells were then fused with NS-1 myeloma cells. PCV3-specific monoclonal antibodies (MAbs) were identified by immunofluorescent antibodies (IFA) using HEK293 cells expressing the native PCV3 cap. HEK293 cells in a 6-well plate grown in MEM medium with 5% fetal bovine serum (FBS) and antibiotics (ciprofloxacin, penicillin, streptomycin and gentamicin) were transfected with pSF-CMV-AMP or a plasmid derived from pSF-CMV- AMP, which contained the complete PCV3 cap gene cloned into the XhoI restriction site (pSF-CMV-cap). In a 6-well plate, 10 ⁶ cells were transfected with Lipofectamine™ 2000 (Invitrogen), according to the manufacturer's instructions, and 10 μg of DNA. After 48 hours, the plates were fixed with 80% acetone for 10 minutes at room temperature (RT) and left to dry. Transfected cells were incubated with undiluted anti-PCV3-cap MAb at 37°C for 1 hour, followed by washing three times with phosphate buffered saline (PBS), then incubated with fluorescein isothiocyanate (FITC)-conjugated goat anti-mouse IgG (Jackson Immunoresearch Laboratories Inc West Grove, PA) diluted 1:100 in PBS at 37°C for one hour. After the final wash, cells were imaged using an Eclipse TE2000-U inverted fluorescence microscope (Nikon). PCV2-infected ST cells were used to evaluate the specificity of MAbs to PCV3.

Example 5

[240] This example demonstrates PCV3 detection and immunohistochemistry in cases of PDNS.

[241] Tissues were fixed in 10% neutral buffered formalin at RT and embedded in paraffin until sectioned. Immunohistochemistry was performed according to a standard protocol, and microscopic slides were imaged using an LSM 700 confocal scanning microscope (Zeiss).

Example 6

[242] This example demonstrates the development of an ELISA for the recombinant PCV3 capsid protein.

[243] The ELISA was performed similarly to a previously described assay using 2 μg/mL of purified recombinant PCV3 capsid protein per well to coat Corning EIA/RIA high-binding plates (33–35).

[244] Results for Examples 1-6

[245] Complete genome sequences of PCV3 used in phylogenetic analyzes have been provided to Genbank under accession numbers KT869077 (PCV3 strain 29160) and KX458235 (PCV3 strain 2164). Previously published sequences used for phylogenetic analysis include: catfish circovirus (Barbel) (BarCV) GU799606; bat circovirus-1 (BtCV-1), JX863737; bat circovirus-2 (BtCV-2) KC339249; bat circovirus-3 (BtCV-3) JQ814849; Beak and Feather Disease Virus (BFDV) AF080560; canary circovirus (CaCV) AJ301633; canine circovirus (CanineCV) KC241983; Chimpanzee fecal associated circovirus (ChfaCV) GQ404851; duck circovirus (DuCV) AY228555; European catfish circovirus (EcatfishCV) JQ011377; finch circovirus (FiCV) DQ845075; goose ciruvirus (GoCV) AJ304456; Gull circuvirus (GuCV) DQ845074; human feces associated circuvirus (HufaCV) GQ404856; mink circovirus (MiCV) KJ020099; Columbid circuvirus (PiCV) AF252610; porcine cirrovirus 1 (PCV1) Y09921; porcine cirrovirus 2 (PCV2) AF027217; PorkNW2/USA/2009 HQ738638; Crow circuvirus (RaCV) DQ146997; starling circuvirus (StCV) DQ172906; swan circovirus (SwCV) EU056309; Zebra finch cyrovirus (ZfiCV) KP793918.

[246] Clinical and histological findings from a study of an outbreak of PDNS-like diseases on a commercial farm.

[247] During June 2015, on a commercial swine farm, sow mortality increased by 10.2% and conceptions decreased by 0.6% compared to historical farm averages due to a PDNS outbreak. According to clinical assessment, the affected sows were anorexic and had multifocal papules, macules and superficial dermatitis. Tissue samples were provided to ISUVDL for diagnostic tests. Histologically, the skin lesions were characterized by acute necrotizing dermatitis and epidermatitis associated with lymphoplasmacytic perivascular cell accumulations. In the kidneys, dilated cortical tubules, weakening and regeneration of the epithelium lining the renal tubules, and large clusters of lymphocytes and macrophages diffusely infiltrated into the cortical interstitium and glomeruli were observed. On the farm, the number of aborted mummified fetuses per litter increased by 1.19 times the historical abortion average. The aborted litters contained mummified fetuses of varying gestational ages consistent with previously described PCV2-associated abortions. While the extensive and histological lesions observed in sows, as well as the presence of abortions, were consistent with PCVAD, all pig tissues, including kidneys, lymph nodes, lungs and skin, were negative by immunohistochemistry (IHC) and qPCR for the presence of PCV2, PRRSV and IAV. In addition, germinal tissues were negative for PCV2, PRRSV, and PPV by qPCR.

[248] Metagenomic sequencing

[249] A pool of tissue homogenates obtained from three fetuses was analyzed by sequencing of viral metagenomes. The Miseq run generated 989,478 reads mapping 926,380 to the Sus scrofa host genome. The remaining reads were clustered de novo , resulting in 27 contigs. Approximately 54% of the reads mapped to 1246 bp. a contig that was 98% similar to the partial circuvirus genome identified in commercial ground pork PorkNW2/USA/2009 (accession number HQ738638), according to the results of BLASTN analysis. The remaining reads showed no similarity to any known eukaryotic virus.

[250] Metagenomic sequencing was also performed on pooled tissue homogenates obtained from tissue samples from sows with PDNS-like lesions. De novo clustering of sequences not included in the mapping relative to Sus scrofa yielded 735 contigs, which were analyzed by BLASTN. The two contigs had approximately 97% identity with hepatitis virus 1 (TTV-1). Clustering using reference PCV3 (KT869077) identified mapping of four reads to the genome. The remaining reads showed no homology to known eukaryotic viruses.

[251] Genetic analysis

[252] Rolling ring amplification followed by PCR and Sanger sequencing of the resulting amplicons made it possible to obtain an assembly of a circular genome consisting of 2000 nucleotides (nt) from a homogenate of germ tissues. ORF analysis revealed three ORFs encoding proteins larger than 200 amino acids (aa), and according to the results of BLASTP analysis, two ORFs showed homology with the circuvirus rep and cap proteins, oriented in opposite directions (Fig. 1). Within the 235 nt 5' intergenic region between the rep and cap ORFs on the rep gene strand there was a predicted stem-loop structure consisting of a 9 nt stem and a nanometer loop identical to that of PCV1 (TAGTATTAC) (SEQ ID No. 15 ). Following convention, the ʺAʺ residue at position 8 in the nanoloop was defined as the ʺ1ʺ position in the genome.

[253] The largest ORF encoded a predicted 297 aa protein, which according to BLASTP analysis was 69.4% identical to the partial replicase protein Circoviridae PorkNW2/USA/2009 (accession number ADU77001, 221 aa) and 54% identical to bat circovirus from China (accession number AIF76248, 293aa). The PorkNW2/USA/2009 genome was obtained from commercial pork meat products and encoded a complete replicase and a partial capsid protein gene, which were found to be most similar in organization and sequence to circuviruses. Conserved circovirus replicase and helicase domains were identified by BLASTP analysis from aa 9-93 and 162-251 regions in the rep ORF, respectively. Further examination of the rep ORF protein sequence revealed conserved rolling circle replication (RCR) and P-loop motifs similar to GoCV and PiCV. Of the three conserved circovirus RCR motifs, the FTLNN motif (SEQ ID No. 20) in PCV3 contained one mutation, represented as FTINN (SEQ ID No. 21). This mutation is observed in other circoviruses such as GoCV. Two other RCR motifs, HLQG (SEQ ID No. 22) and YCKK (SEQ ID No. 23), are also present in PCV3. In addition, three conserved motifs in circovirus replicase proteins of unknown function have been identified in PCV3, including WWDGY (amino acids 196-200) (SEQ ID No. 24), DDFYGWVP (amino acids 209-216) (SEQ ID No. 25), and DRYP (amino acids 225-228) (SEQ ID No. 26). Interestingly, the canonical initiation codon has not been identified. The GTC (encoding valine) codon (SEQ ID No. 27) is present at the 5' end of the ORF with the nearest ATG (SEQ ID No. 28) in frame located approximately 400 bp downstream. This alternative start codon was also found in PorkNW2/USA/2009. Alternative start codons have been proposed for several avian circoviruses, including goose circovirus, pigeon circovirus, and beak and feather disease virus.

[254] According to the results of BLASTP analysis, the putative cap ORF with the opposite orientation relative to rep encodes a 214 aa protein with 87% identity to the partial sequence of the capsid protein (110 aa) PorkNW2/USA/2009 and 36-37% identity to PCV2 and duck circoviruses ( 233 and 257 aa, respectively) (Fig. 1). Like other circovirus capsid proteins, the N-terminus contained numerous arginine residues and was highly basic. The conserved domain of the circovirus capsid protein was identified by BLASTP analysis from aa 26-173. In addition, the PCV3 cap protein did not have predicted N-linked glycosylation sites, but contained two predicted O-linked glycosylation sites at positions 146 and 150 aa (S and T, respectively). This is in contrast to PCV2, which has two experimentally confirmed N-linked glycosylation sites.

[255] A third ORF, located on the same strand as the predicted rep , encodes a 231 aa protein that is 94% identical to the ORF identified in PorkNW2/USA/2009 and 39% identical to murine herpesvirus M169, a protein of unknown function . Similar to rep, the initiation codon for ORF3 is unclear. The codon at the 5' end is TCG (coding serine). The methionine at position 55 aa of ORF3 represents an alternative possible initiation site that gives rise to the 177 aa protein.

[256] Due to genetic and structural similarity to the genus Circovirus and <70% capsid aa identity with other species, the new species is proposed as porcine circovirus type 3 (PCV3). The PCV3 genome sequences have been submitted to Genbank with accession numbers KT869077 and KX458235.

[257] Phylogenetic analysis

[258] To study the evolutionary relationship of PCV3 with other members of the family Circoviridae , the genome sequences of twenty-three family members and two PCV3 genomes were analyzed. Analysis of complete circovirus genomes allowed both PCV3 sequences to be grouped in a clade with PorkNW2/USA/2009, separate from all other members of the genus (Fig. 2). Phylogenetic analysis showed that PCV3 is most closely related to canine circovirus (KC241983), but this relationship lacks strong bootstrap support. Phylogenetic analysis also suggests that PCV3 and canine CV share a common ancestor with the clade containing PCV1, PCV2, and BatCV-2 (KC339249). With the exception of human circuvirus (HufaCV, GQ404856), mammalian and avian cirroviruses belong to distinct, well-confirmed clades.

[259] Detection of PCV3 by PCR

[260] To confirm the presence of PCV3 in pig samples, a 5' nuclease assay was developed to detect the presence of the PCV3 cap gene. Germ tissue homogenate samples obtained from an outbreak farm were strongly positive for PCV3 with Ct values between 16.7 and 21.3, corresponding to high PCV3 levels of approximately 1.88 x 10 ⁸ and 7.55 x 10 ⁶ g. (genomic copies)/ml. Tissues from three sows with PDNS-like lesions were positive for PCV3, ranging from 2.13 x 10 ⁴ to 8.62 x 10 ⁴ kg/ml. In addition, 30 serum samples submitted to ISUVDL were analyzed for the presence of PCV3 by qPCR. Serum samples were positive for PCV3 with a value of 5.63×10 ² -2.28×10 ⁴ gc/ml. The serum sample with the highest PCV3 titer was used for PCR amplification to generate overlapping amplicons to obtain a second complete PCV3 genome that was found to be 99.0% identical to the original PCV3 from the outbreak farm. This second PCV3 genome has been submitted to Genbank with accession number KX458235.

[261] Additionally, to study the prevalence of PCV3, a total of 271 samples submitted to ISUVDL for respiratory diagnostic testing by qPCR were analyzed. Thirty-four (12.5%) samples had positive titers: 3.00×10 ² -1.52×10 ⁷ gc/ml.

[262] Characteristics of MAb14 to PCV3- cap

[263] HEK293 cells transfected with pSF-CMV-cap were incubated separately with four different clonal cell MAb supernatants and screened by IFA. Nuclear-localized fluorescence, as expected from the predicted highly basic nuclear localization signal, was observed for clone 14 (MAb14). No fluorescence was observed for cells transfected with pSF-CMV-Amp. In addition, no fluorescence was observed in PCV2-infected ST cells.

[264] Virus isolation

[265] The virus was isolated from ST and PK-15 cells. The cells were inoculated with filtered germ tissue homogenates and passaged three times. No cytopathic effects were observed, and Ct values increased with each subsequent passage. IFA showed no fluorescence when MAb14 was used.

[266] Histological lesions associated with the presence of PCV3 antigen in cases of PDNS

[267] Tissue samples from sows with PDNS-like lesions and archival cases of PDNS were studied by H&E staining and IHC using anti-PCV3 MAb14. The lungs showed a variable degree of bronchointerstitial pneumonia, sometimes complicated by secondary purulent bronchopneumonia. Small and medium-sized airways and small blood vessels were clogged with peribronchiolar and perivascular aggregates of lymphocytes and plasma cells. Adjacent alveolar septa were infiltrated with lymphocytes and plasma cells. There was severe intraluminal edema in the alveolar lumen, mixed with moderate numbers of foamy macrophages, rare multinucleated giant cells, and small clusters of neutrophils. Incidental lymphocytes and scattered macrophages gave moderate intracytoplasmic immunostaining against PCV3. In the skin area, the dermis and subcutaneous layer had characteristic necrotizing vasculitis with fibrinoid changes and transmural infiltration of neutrophils, hemorrhage and fibrin exudation. The inflammatory infiltrate in many cases extends to the surrounding dermis and subcutaneous layer. Scattered lymphoplasmacytic aggregates, which sometimes surrounded the vessels, and cutaneous adnexa were also found. In some cases, hyperplastic epidermis with mild orthokeratotic hyperkeratosis was observed. The cutaneous lymphocytic infiltration gave prominent intracytoplasmic immunostaining against PCV3. Minimal background staining was evident when PCV3MAb14 was replaced with PBS.

[268] Diffuse granulomatous lymphadenitis was found in the lymph nodes. Cortical follicles showed mild lymphoid depletion and infiltrated histiocytes and numerous multinucleated giant cells. The subcortical and medullary sinuses were dilated due to the presence of a moderate amount of edema and hemorrhage mixed with numerous macrophages and plasma cells. Follicular and perifollicular lymphocyte populations showed diffuse, intense intracytoplasmic anti-PCV3 staining compared with background staining where PCV3 MAb14 was replaced with PBS, or compared with lymph node tissue obtained from a pig negative for PCV3 by qPCR.

[269] Kidney sections were characterized by the presence of diffuse membranous proliferative glomerulonephritis. Severe glomerulosclerosis was observed, Bowman's capsules were often thickened, and the cortical tubules were weakened and associated with varying degrees of interstitial fibrosis. The glomerular mesangium was hypercellular and thickened with amorphous eosinophilic material. In some cases, the tubules were ectatic, lined with thin epithelium and sometimes with pronounced proteosis. Scattered small to medium sized clusters of lymphocytes and plasma cells in the interstitium were observed in all sections. Occasional areas of positive staining for PCV3 were observed in the tubular epithelium. Minimal background fluorescence was observed in PBS and goat anti-mouse FITC mock slides or kidney tissue obtained from a pig negative for PCV3 by qPCR.

[270] Detection of PCV3 nucleic acid in tissues with PDNS lesions

[271] To further explore the etiological role of PCV3 in PDNS, 48 cases of histological lesions consistent with PDNS that were previously tested negative for PCV2 by IHC (immunohistochemistry) were evaluated. Tissue samples from paraffin-embedded tissue blocks were analyzed for the presence of PCV3 by qPCR. Forty-five (93.8%) cases were positive for PCV3 with viral titers of 1.60-3.47×10 ⁴ gc/ml. To confirm these results, the five samples with the highest viral titers were analyzed by PCR targeting 330 bp. cap gene fragment. The target amplicon was amplified in all samples, and the sequences of the amplicon products showed 100% identity to the PCV3 genome sequence. Tissue from five PCV3 PCR-positive cases was tested by IHC for the presence of PCV3. Three out of five were positive.

[272] PCV3 serotype dominance

[273] The prevalence of antibodies to cap PCV3 in pig sera was studied by ELISA using the PCV3- cap antigen. Eighteen serum samples from 3-week-old pigs obtained from a pathogen-specific herd that were negative for PCV3 by qPCR were used as negative controls with a mean absorbance value of 0.49. The cutoff value differentiating positive and negative sera was defined as three standard deviations from the mean of the negative controls (0.87). Sera from ten sows from an outbreak farm collected three months after the PDNS outbreak were positive with a mean absorbance value of 1.27. In addition, 27 sera obtained from young pigs from a farm that supplies animals to sow replacement farms were also tested, with seventeen animals (63%) having absorbances of 0.88-1.37. Anti-PCV3- cap antibodies were detected in 47 of 83 (56.6%) specimens submitted for independent diagnostic testing from multiple states. Of the positive samples, 13 were from Iowa, one from Indiana, five from Mexico, four from North Carolina, five from Nebraska, one from Oklahoma and 18 were of unknown origin.

[274] Discussion

[275] First described in 1993 in Europe, PDNS has been registered in many countries around the world. Although the prevalence of the disease in a herd is usually low (<1%), mortality in affected pigs can be high. The incidence of PDNS may exceed the incidence of PMWS in Europe and the United Kingdom. Although the etiology of PDNS is unknown, PCV2 nucleic acid is usually detected in affected pigs by qPCR, while PCV2 antigen is not detected in all cases. This has led to the suggestion of a role for PCV2 in PDNS. The present invention has enabled the detection of a highly divergent new species of porcine circovirus, designated PCV3, in samples of mummified aborted fetuses from sows with PDNS-like lesions, and samples obtained from sows that died as a result of acute infection with clinical signs consistent with PDNS. PCV3 was the only virus identified by metagenomic sequencing of pooled germ tissue samples, which was confirmed by qPCR results. Threshold cycle values of 16.7-21.3 in germinal tissue pools show high viral titers of 7.55×10 ⁶ -1.88×10 ⁸ gc/ml sample. There is a correlation between PCV2 titer in fetuses and reproductive disease, with PCV2 levels of 10 ⁷ copies of PCV2 DNA/500 ng of germ tissue or higher being associated with PCV2-associated reproductive failure, including mummification. Based on the amount of PCV3 nucleic acids and tissue distribution detected during this outbreak, a similar correlation was found for PCV3 and reproductive failure.

[276] PCV3 has also been detected by PCR and IHC in the skin, lungs, kidneys and lymph nodes of sows with PDNS-like lesions. Both PCR, IHC and metagenomic sequencing failed to identify PCV2 in samples collected from the outbreak farm. These results indicate that PCV3 infection contributes to PDNS-like lesions, abortive cases, and PCV3 in fetuses are the result of vertical transmission. In support of this etiological role of PCV3 in PDNS lesions, screening of archival PDNS cases negative for PCN2 by IHC was performed, which showed that PCV3 nucleic acid was highly abundant (93.8%), and three of five cases tested by IHC were PCV3 were positive. It should be noted that relatively low titers of PCV3 in tissues may hinder reliable detection.

[277] Attempts to experimentally reproduce PDNS using PCV2 were unsuccessful, but PDNS was experimentally reproduced in the absence of PCV2 using PRRSV and tissue homogenate containing TTV. Relatively little is known about the clinical significance of TTV infection. TTV occurs in pigs throughout the world. While TTV is usually found in healthy pigs, several studies have suggested that TTV infection slows the progression of the disease to severe disease during coinfection. For example, inoculation of pigs with tissue homogenate containing TTV followed by infection with PCV2 led to the development of PMWS, whereas this was not observed with monoinfection. Sows from a farm with a PDNS outbreak were infected with both PCV3 and TTV1. The impact of co-infection with TTV1 is unknown. It is unclear whether co-infection with genetically diverse small circular DNA viruses such as TTV1 ( Anelloviridae ), PCV2 and PCV3 influences the development of PDNS. Additive effects on disease severity caused by PCV2 co-infections have been demonstrated for PMWS. Co-infection with PPV and PCV2 showed that the disease was worsening. Co-infections with PCV2 and PRRSV are a significant component of the porcine respiratory disease complex. Infection of herds with these agents causes severe respiratory disease and economically devastating abortions and mortality.

[278] The pathogenesis of PDNS, which includes a characteristic necrotizing vasculitis, is thought to be a manifestation of an immune complex-mediated disorder involving PCV2. In the control study, all pigs with clinical signs of PDNS were positive for PCV2 by PCR and had significantly higher PCV2 antibody titers than clinically normal pigs. A study of the kidneys of PDNS positive pigs revealed an increase in fibrinoid deposits in the glomeruli, consisting of accumulated IgG1, IgG2, IgM, and complement factors C1q and C3, compared with clinically normal pigs. Although PCV2 antigen was identified by IHC in the lung tissue of these PDNS pigs, PCV2 antigen was not identified in immune complexes. This result is similar to the inconsistent detection of PCV2 in porcine PDNS kidney tissues reported elsewhere. Viral infections are known to contribute to immunological disorders due to which Aleutian mink disease (AMD), caused by Aleutian mink disease virus (AMDV), has a similar pathogenesis to PDNS. The pathogenesis of AMD is associated with the overproduction of AMDV-specific IgG antibodies. The role of PCV3 in a possible immune complex-mediated disorder leading to the pathogenesis of PDNS needs further investigation.

[279] PCV2 is one of the most economically important viral pathogens of pigs worldwide. The most common genotypes associated with PCVAD are PCV2a and PCV2b. Until 2003, PCV2a was the main genotype identified in the United States and Canada, although both PCV2a and PCV2b have been found throughout the world. Around 2003, there was a dramatic shift in the frequency of PCV2 genotypes globally from PCV2a to PCV2b, coinciding with severe systemic disease associated with PCV2b. The worldwide epidemic has been successfully controlled by the development of commercial vaccines that contain the PCV2a antigen, which have been shown to be cross-protective. More recently, a new genotype, PCV2d, first discovered in Switzerland in 1999, spread to China and then to the United States. As with PCV2b, epidemiological studies suggest that a genotype shift is currently occurring, as evidenced by reports of PCV2 vaccine failure and an increase in clinical cases of the disease. PCV2d has been shown to be involved in the development of more severe clinical signs and lesions.

[280] Circoviruses are genetically diverse and infect a wide range of hosts through documented cross-species transmission. Phylogenetic analysis suggests the closest evolutionary relationship between PCV3 and CanineCV. Interestingly, CanineCV was identified in the liver of a dog with severe necrotizing vasculitis and granulomatous lymphadenitis, both of which were observed in PCV3-infected pigs, and PCV2 infections have also been reported. It is unclear whether PCV3 has evolved as an undetected infection in pigs for some time, or whether it has arisen through cross-transmission or recombination between unidentified parental circoviruses. PCV2 is capable of crossing species barriers to cause fatal diseases in species other than pigs; a recent report showed the presence of PCV2 in six minks that died of diarrhea in China.

[281] The discovery of a novel porcine circuvirus with a likely etiological role in PDNS and reproductive failure is concerning. Retrospective studies indicate that PCV2 caused sporadic systemic disease earlier in 1985 before an epidemic emerged in the late 1990s. The possibility that PCV3 develops in a similar manner merits further study and highlights the need for the production of immunogenic compositions of the present invention. It should be noted that given that the capsid proteins of PCV2 and PCV3 are approximately 30% identical, cross-protection seems unlikely.

Conclusion

[282] Porcine circovirus 2 is one of the most important pathogens of pigs worldwide. This application demonstrates the discovery of a highly divergent new species of porcine circovirus PCV3 in sows with aborted fetuses and signs of PDNS, the clinical manifestation of which is usually associated with PCV2 infection. The PCV3 genome includes ORF1, ORF2 and ORF3. No other viruses were detected by metagenomic sequencing, and PCR results were negative for PCV2, PPV, PRRSV, and IAV. This, coupled with the high viral load in mummies' tissue, suggests that PCV3 may cause clinical disease similar to PCV2. Molecular and serological analyzes for PCV3 also suggest that the virus is a common virus circulating in US swine herds. Because PCV2 infection did not initially manifest clinically and only sporadically caused disease before becoming a global epidemic, PCV3 requires further study.

The contents of the following documents are incorporated herein by reference.

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[286] Meehan BM, McNeilly F, McNair I, Walker I, Ellis JA, Krakowka S, et al. Isolation and characterization of porcine circovirus 2 from cases of sow abortion and porcine dermatitis and nephropathy syndrome. Arch Virol. 2001; 146:835-842

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[288] Cortey M, Pileri E, Sibila M, Pujols J, Balasch M, Plana J, et al. Genotypic shift of porcine circovirus type 2 from PCV-2a to PCV-2b in Spain from 1985 to 2008. Vet J. 2011; 187:363-8.

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--->

LIST OF SEQUENCES

<110> KANSAS STATE UNIVERSITY RESEARCH FOUNDATION

House, Ben

<120> IMMUNOGENIC COMPOSITIONS FOR IMMUNIZATION OF PIGS AGAINST

CIRCOVIRUS TYPE 3 AND METHODS OF THEIR OBTAINING AND APPLICATION

<130> 40405-PRO

<160> 28

<170> Patent In version 3.5

<210> 1

<211> 2000

<212> DNA

<213> Porcine circovirus 3

<400> 1

acccggcacc tcggaacccg gatccacgga ggtctgtagg gagaaaaagt ggtatcccat 60

tatggatgct ccgcaccgtg tgagtggata taccggggcag tggatgatga agcggcctcg 120

tgttttgatg ccgcaggacg gggactggat aactgagttt ttgtggtgct acgagtgtcc 180

tgaagataag gacttttatt gtcatcctat tctaggtccg gagggaaagc ccgaaacaca 240

ggtggtgttt tacgataaac aactggaccc cgaccgagtg ggaatctatt gtggagtgtg 300

gaggcagtat agcgagatac cttattatcg gcaaagaggt tggaaaaagc ggtaccccac 360

acttgcaagg gtacgtgaat ttcaagaaca aaaggcgact cagctcggtg aagcgcttac 420

ccggatttgg tcgggcccat ctggagccgg cgagggggag ccacaaagag gccagcgagt 480

attgcaagaa agagggggat tacctcgaga ttggcgaaga ttcctcttcg ggtaccagat 540

cggatcttca agcagcagct cggattctga cggagacgtc gggaaatctg actgaagttg 600

cggagaagat gcctgcagta tttatacgct atgggcgggg tttgcgtgat ttttgcgggg 660

tgatggggtt gggtaaaccg cgtgatttta aaactgaagt ttatgttttt attggtcctc 720

caggatgcgg gaaaacgcgg gaagcttgtg cggatgcggc tgcgcgggaa ttgcagttgt 780

atttcaagcc acggggggcct tggtgggatg gttataatgg ggagggtgct gttattttgg 840

atgattttta tgggtgggtt ccatttgatg aattgctgag aattggggac aggtaccctc 900

tgagggttcc tgttaagggt gggtttgtta attttgtggc taaggtatta tatattacta 960

gtaatgttgt accggaggag tggtattcat cggagaatat tcgtggaaag ttggaggcct 1020

tgtttaggag gttcactaag gttgtttgtt ggggggaggg gggggtaaag aaagacatgg 1080

agacagtgta tccaataaac tattgatttt atttgcactt gtgtacaatt attgcgttgg 1140

ggtggggggta tttattggga gggtgggtgg gcagccccct agccacggct tgtcgccccc 1200

accgaagcat gtgggggatg gggtccccac atgcgagggc gtttacctgt gcccgcaccc 1260

gaagcgcagc gggagcgcgc gcgaggggac acggcttgtc gccaccggag gggtcagatt 1320

tatatttatt ttcacttaga gaacggactt gtaacgaatc caaacttctt tggtgccgta 1380

gaagtctgtc attccagttt tttccgggac ataaatgctc caaagcagtg ctccccattg 1440

aacggtgggg tcatatgtgt tgagccatgg ggtgggtctg gagaaaaaga agaggctttg 1500

tcctgggtga gcgctggtag ttcccgccag aagtggtttg ggggtgaagt aacggctgtg 1560

ttttttttta gaagtcataa ctttacgagt ggaactttcc gcataagggt cgtcttggag 1620

ccaagtgttt gtggtccagg cgccgtctag atctatggct gtgtgcccga acatagtttt 1680

tgtttgctga gccggagaaa ttacagggct gagtgtaact ttcattttta gtatcttata 1740

atattcaaag gtaattgcag tttcccattc gtttaggcgg gtaatgaagt ggttggcgtg 1800

ccagggcttg ttattctgag gggttccaac ggatatgacg ttcattgtgg agtatttctt 1860

tgtgtagtat gtgccagctg tgggcctcct aatgaatagt cgtcttctgg catagcgcct 1920

tctgtggcgt cgtcgtctcc ttgggcgggg tcttcttctg aatatagctc tgtgtctcat 1980

tttggtgccg ggctagtatt 2000

<210> 2

<211> 646

<212> protein

<213> Porcine circovirus

<400> 2

Thr Arg His Leu Gly Thr Arg Ile His Gly Gly Leu Gly Glu Lys Val

1 5 10 15

Val Ser His Tyr Gly Cys Ser Ala Pro Cys Glu Trp Ile Tyr Arg Ala

20 25 30

Val Asp Asp Glu Ala Ala Ser Cys Phe Asp Ala Ala Gly Arg Gly Leu

35 40 45

Asp Asn Val Phe Val Val Leu Arg Val Ser Arg Gly Leu Leu Leu Ser

50 55 60

Ser Tyr Ser Arg Ser Gly Gly Lys Ala Arg Asn Thr Gly Gly Val Leu

65 70 75 80

Arg Thr Thr Gly Pro Arg Pro Ser Gly Asn Leu Leu Trp Ser Val Glu

85 90 95

Ala Val Arg Asp Thr Leu Leu Ser Ala Lys Arg Leu Glu Lys Ala Val

100 105 110

Pro His Thr Cys Lys Gly Thr Ile Ser Arg Thr Lys Gly Asp Ser Ala

115 120 125

Arg Ser Ala Tyr Pro Asp Leu Val Gly Pro Ile Trp Ser Arg Arg Gly

130 135 140

Gly Ala Thr Lys Arg Pro Ala Ser Ile Ala Arg Lys Arg Gly Ile Thr

145 150 155 160

Ser Arg Leu Ala Lys Ile Pro Leu Arg Val Pro Asp Arg Ile Phe Lys

165 170 175

Gln Gln Leu Gly Phe Arg Arg Arg Arg Glu Ile Leu Lys Leu Arg Arg

180 185 190

Arg Cys Leu Gln Tyr Leu Tyr Ala Met Gly Gly Val Cys Val Ile Phe

195 200 205

Ala Gly Trp Gly Trp Val Asn Arg Val Ile Leu Lys Leu Lys Phe Met

210 215 220

Phe Leu Leu Val Leu Gln Asp Ala Gly Lys Arg Gly Lys Leu Val Arg

225 230 235 240

Met Arg Leu Arg Gly Asn Cys Ser Cys Ile Ser Ser His Gly Gly Leu

245 250 255

Gly Gly Met Val Ile Met Gly Arg Val Leu Leu Phe Trp Met Ile Phe

260 265 270

Met Gly Gly Phe His Leu Met Asn Cys Glu Leu Gly Thr Gly Thr Leu

275 280 285

Gly Phe Leu Leu Arg Val Gly Leu Leu Ile Leu Trp Leu Arg Tyr Tyr

290 295 300

Ile Leu Leu Val Met Leu Tyr Arg Arg Ser Gly Ile His Arg Arg Ile

305 310 315 320

Phe Val Glu Ser Trp Arg Pro Cys Leu Gly Gly Ser Leu Arg Leu Phe

325 330 335

Val Gly Gly Arg Gly Gly Arg Lys Thr Trp Arg Gln Cys Ile Gln Thr

340 345 350

Ile Asp Phe Ile Cys Thr Cys Val Gln Leu Leu Arg Trp Gly Gly Gly

355 360 365

Ile Tyr Trp Glu Gly Gly Trp Ala Ala Pro Pro Arg Leu Val Ala Pro

370 375 380

Thr Glu Ala Cys Gly Gly Trp Gly Pro His Met Arg Gly Arg Leu Pro

385 390 395 400

Val Pro Ala Pro Glu Ala Gln Arg Glu Arg Ala Arg Gly Asp Thr Ala

405 410 415

Cys Arg His Arg Arg Gly Gln Ile Tyr Ile Tyr Phe His Leu Glu Asn

420 425 430

Gly Leu Val Thr Asn Pro Asn Phe Phe Gly Ala Val Glu Val Cys His

435 440 445

Ser Ser Phe Phe Arg Asp Ile Asn Ala Pro Lys Gln Cys Ser Pro Leu

450 455 460

Asn Gly Gly Val Ile Cys Val Glu Pro Trp Gly Gly Ser Gly Glu Lys

465 470 475 480

Glu Glu Ala Leu Ser Trp Val Ser Ala Gly Ser Ser Arg Gln Lys Trp

485 490 495

Phe Gly Gly Glu Val Thr Ala Val Phe Phe Phe Arg Ser His Asn Phe

500 505 510

Thr Ser Gly Thr Phe Arg Ile Arg Val Val Leu Glu Pro Ser Val Cys

515 520 525

Gly Pro Gly Ala Val Ile Tyr Gly Cys Val Pro Glu His Ser Phe Cys

530 535 540

Leu Leu Ser Arg Arg Asn Tyr Arg Ala Glu Cys Asn Phe His Phe Tyr

545 550 555 560

Leu Ile Ile Phe Lys Gly Asn Cys Ser Phe Pro Phe Val Ala Gly Asn

565 570 575

Glu Val Val Gly Val Pro Gly Leu Val Ile Leu Arg Gly Ser Asn Gly

580 585 590

Tyr Asp Val His Cys Gly Val Phe Leu Cys Val Val Cys Ala Ser Cys

595 600 605

Gly Pro Pro Asn Glu Ser Ser Ser Gly Ile Ala Pro Ser Val Ala Ser

610 615 620

Ser Ser Pro Trp Ala Gly Ser Ser Ser Glu Tyr Ser Ser Val Ser His

625 630 635 640

Phe Gly Ala Gly Leu Val

645

<210> 3

<211> 891

<212> DNA

<213> Porcine circovirus ORF1

<400> 3

gtccggaggg aaagcccgaa acacaggtgg tgttttacga taaacaactg gaccccgacc 60

gagtgggaat ctattgtgga gtgtggaggc agtatagcga gataccttat tatcggcaaa 120

gaggttggaa aaagcggtac cccacacttg caagggtacg tgaatttcaa gaacaaaagg 180

cgactcagct cggtgaagcg cttacccgga tttggtcggg cccatctgga gccggcgagg 240

gggagccaca aagaggccag cgagtattgc aagaaagagg gggattacct cgagattggc 300

gaagattcct cttcgggtac cagatcggat cttcaagcag cagctcggat tctgacggag 360

acgtcgggaa atctgactga agttgcggag aagatgcctg cagtatttat acgctatggg 420

cggggtttgc gtgatttttg cggggtgatg gggttgggta aaccgcgtga ttttaaaact 480

gaagtttatg tttttattgg tcctccagga tgcgggaaaa cgcgggaagc ttgtgcggat 540

gcggctgcgc gggaattgca gttgtatttc aagccacggg ggccttggtg ggatggttat 600

aatggggagg gtgctgttat tttggatgat ttttatgggt gggttccatt tgatgaattg 660

ctgagaattg gggacaggta ccctctgagg gttcctgtta agggtggggtt tgttaatttt 720

gtggctaagg tattatatat tactagtaat gttgtaccgg aggagtggta ttcatcggag 780

aatattcgtg gaaagttgga ggccttgttt aggaggttca ctaaggttgt ttgttggggg 840

gagggggggg taaagaaaga catggagaca gtgtatccaa taaactattg a 891

<210> 4

<211> 296

<212> protein

<213> Porcine circovirus ORF1

<400> 4

Val Arg Arg Glu Ser Pro Lys His Arg Trp Cys Phe Thr Ile Asn Asn

1 5 10 15

Trp Thr Pro Thr Glu Trp Glu Ser Ile Val Glu Cys Gly Gly Ser Ile

20 25 30

Ala Arg Tyr Leu Ile Ile Gly Lys Glu Val Gly Lys Ser Gly Thr Pro

35 40 45

His Leu Gln Gly Tyr Val Asn Phe Lys Asn Lys Arg Arg Leu Ser Ser

50 55 60

Val Lys Arg Leu Pro Gly Phe Gly Arg Ala His Leu Glu Pro Ala Arg

65 70 75 80

Gly Ser His Lys Glu Ala Ser Glu Tyr Cys Lys Lys Glu Gly Asp Tyr

85 90 95

Leu Glu Ile Gly Glu Asp Ser Ser Ser Gly Thr Arg Ser Asp Leu Gln

100 105 110

Ala Ala Ala Arg Ile Leu Thr Glu Thr Ser Gly Asn Leu Thr Glu Val

115 120 125

Ala Glu Lys Met Pro Ala Val Phe Ile Arg Tyr Gly Arg Gly Leu Arg

130 135 140

Asp Phe Cys Gly Val Met Gly Leu Gly Lys Pro Arg Asp Phe Lys Thr

145 150 155 160

Glu Val Tyr Val Phe Ile Gly Pro Pro Gly Cys Gly Lys Thr Arg Glu

165 170 175

Ala Cys Ala Asp Ala Ala Ala Arg Glu Leu Gln Leu Tyr Phe Lys Pro

180 185 190

Arg Gly Pro Trp Trp Asp Gly Tyr Asn Gly Glu Gly Ala Val Ile Leu

195 200 205

Asp Asp Phe Tyr Gly Trp Val Pro Phe Asp Glu Leu Leu Arg Ile Gly

210 215 220

Asp Arg Tyr Pro Leu Arg Val Pro Val Lys Gly Gly Phe Val Asn Phe

225 230 235 240

Val Ala Lys Val Leu Tyr Ile Thr Ser Asn Val Val Pro Glu Glu Trp

245 250 255

Tyr Ser Ser Glu Asn Ile Arg Gly Lys Leu Glu Ala Leu Phe Arg Arg

260 265 270

Phe Thr Lys Val Val Cys Trp Gly Glu Gly Gly Val Lys Lys Asp Met

275 280 285

Glu Thr Val Tyr Pro Ile Asn Tyr

290 295

<210> 5

<211> 645

<212> DNA

<213> Porcine circovirus ORF2

<400> 5

atgagacaca gagctatatt cagaagaaga ccccgcccaa ggagacgacg acgccacaga 60

aggcgctatg ccagaagacg actattcatt aggaggccca cagctggcac atactacaca 120

aagaaatact ccacaatgaa cgtcatatcc gttggaaccc ctcagaataa caagccctgg 180

cacgccaacc acttcattac ccgcctaaac gaatgggaaa ctgcaattac ctttgaatat 240

tataagatac taaaaatgaa agttacactc agccctgtaa tttctccggc tcagcaaaca 300

aaaactatgt tcgggcacac agccatagat ctagacggcg cctggaccac aaacacttgg 360

ctccaagacg acccttatgc ggaaagttcc actcgtaaag ttatgacttc taaaaaaaaa 420

cacagccgtt acttcacccc caaaccactt ctggcgggaa ctaccagcgc tcacccagga 480

caaagcctct tctttttctc cagacccacc ccatggctca acacatatga ccccaccgtt 540

caatggggag cactgctttg gagcatttat gtcccggaaa aaactggaat gacagacttc 600

tacggcacca aagaagtttg gattcgttac aagtccgttc tctaa 645

<210> 6

<211> 214

<212> protein

<213> Porcine circovirus ORF2

<400> 6

Met Arg His Arg Ala Ile Phe Arg Arg Arg Pro Arg Pro Arg Arg Arg

1 5 10 15

Arg Arg His Arg Arg Arg Tyr Ala Arg Arg Arg Leu Phe Ile Arg Arg

20 25 30

Pro Thr Ala Gly Thr Tyr Tyr Thr Lys Lys Tyr Ser Thr Met Asn Val

35 40 45

Ile Ser Val Gly Thr Pro Gln Asn Asn Lys Pro Trp His Ala Asn His

50 55 60

Phe Ile Thr Arg Leu Asn Glu Trp Glu Thr Ala Ile Thr Phe Glu Tyr

65 70 75 80

Tyr Lys Ile Leu Lys Met Lys Val Thr Leu Ser Pro Val Ile Ser Pro

85 90 95

Ala Gln Gln Thr Lys Thr Met Phe Gly His Thr Ala Ile Asp Leu Asp

100 105 110

Gly Ala Trp Thr Thr Asn Thr Trp Leu Gln Asp Asp Pro Tyr Ala Glu

115 120 125

Ser Ser Thr Arg Lys Val Met Thr Ser Lys Lys Lys His Ser Arg Tyr

130 135 140

Phe Thr Pro Lys Pro Leu Leu Ala Gly Thr Thr Ser Ala His Pro Gly

145 150 155 160

Gln Ser Leu Phe Phe Phe Ser Arg Pro Thr Pro Trp Leu Asn Thr Tyr

165 170 175

Asp Pro Thr Val Gln Trp Gly Ala Leu Leu Trp Ser Ile Tyr Val Pro

180 185 190

Glu Lys Thr Gly Met Thr Asp Phe Tyr Gly Thr Lys Glu Val Trp Ile

195 200 205

Arg Tyr Lys Ser Val Leu

210

<210> 7

<211> 696

<212> DNA

<213> Porcine circovirus ORF3

<400> 7

tcgtcttctg gcatagcgcc ttctgtggcg tcgtcgtctc cttgggcggg gtcttcttct 60

gaatatagct ctgtgtctca ttttggtgcc gggctagtat tacccggcac ctcggaaccc 120

ggatccacgg aggtctgtag ggagaaaaag tggtatccca ttatggatgc tccgcaccgt 180

gtgagtggat ataccggggca gtggatgatg aagcggcctc gtgttttgat gccgcaggac 240

ggggactgga taactgagtt tttgtggtgc tacgagtgtc ctgaagataa ggacttttat 300

tgtcatccta ttctaggtcc ggagggaaag cccgaaacac aggtggtgtt ttacgataaa 360

caactggacc ccgaccgagt gggaatctat tgtggagtgt ggaggcagta tagcgagata 420

ccttattatc ggcaaagagg ttggaaaaag cggtacccca cacttgcaag ggtacgtgaa 480

tttcaagaac aaaaggcgac tcagctcggt gaagcgctta cccggatttg gtcgggccca 540

tctggagccg gcgaggggga gccacaaaga ggccagcgag tattgcaaga aagaggggga 600

ttacctcgag attggcgaag attcctcttc gggtaccaga tcggatcttc aagcagcagc 660

tcggattctg acggagacgt cgggaaatct gactga 696

<210> 8

<211> 231

<212> protein

<213> Porcine circovirus ORF3

<400> 8

Ser Ser Ser Gly Ile Ala Pro Ser Val Ala Ser Ser Ser Pro Trp Ala

1 5 10 15

Gly Ser Ser Ser Glu Tyr Ser Ser Val Ser His Phe Gly Ala Gly Leu

20 25 30

Val Leu Pro Gly Thr Ser Glu Pro Gly Ser Thr Glu Val Cys Arg Glu

35 40 45

Lys Lys Trp Tyr Pro Ile Met Asp Ala Pro His Arg Val Ser Gly Tyr

50 55 60

Thr Gly Gln Trp Met Met Lys Arg Pro Arg Val Leu Met Pro Gln Asp

65 70 75 80

Gly Asp Trp Ile Thr Glu Phe Leu Trp Cys Tyr Glu Cys Pro Glu Asp

85 90 95

Lys Asp Phe Tyr Cys His Pro Ile Leu Gly Pro Glu Gly Lys Pro Glu

100 105 110

Thr Gln Val Val Phe Tyr Asp Lys Gln Leu Asp Pro Asp Arg Val Gly

115 120 125

Ile Tyr Cys Gly Val Trp Arg Gln Tyr Ser Glu Ile Pro Tyr Tyr Arg

130 135 140

Gln Arg Gly Trp Lys Lys Arg Tyr Pro Thr Leu Ala Arg Val Arg Glu

145 150 155 160

Phe Gln Glu Gln Lys Ala Thr Gln Leu Gly Glu Ala Leu Thr Arg Ile

165 170 175

Trp Ser Gly Pro Ser Gly Ala Gly Glu Gly Glu Pro Gln Arg Gly Gln

180 185 190

Arg Val Leu Gln Glu Arg Gly Gly Leu Pro Arg Asp Trp Arg Arg Phe

195 200 205

Leu Phe Gly Tyr Gln Ile Gly Ser Ser Ser Ser Ser Ser Asp Ser Asp

210 215 220

Gly Asp Val Gly Lys Ser Asp

225 230

<210> 9

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> PCV3 replicase primer

<400> 9

aggagtggta ttcatcggag a 21

<210> 10

<211> 22

<212> DNA

<213> Artificial sequence

<220>

<223> PCV3 replicase primer

<400> 10

aaacaacctt agtgaacctc ct 22

<210> 11

<211> 23

<212> DNA

<213> Artificial sequence

<220>

<223> PCV3 replicase probe 5' HEX/ZEN/3' IBFQ

<400> 11

acaaggcctc caactttcca cga 23

<210> 12

<211> 19

<212> DNA

<213> Artificial sequence

<220>

<223> PCV3 capsid primer

<400> 12

acacagccgt tacttcacc 19

<210> 13

<211> 18

<212> DNA

<213> Artificial sequence

<220>

<223> PCV3 capsid primer

<400> 13

agtgctcccc attgaacg 18

<210> 14

<211> 25

<212> DNA

<213> Artificial sequence

<220>

<223> capsid probe PCV3 5' 6-FAM/ZEN/3' IBFQ

<400> 14

accccatggc tcaacacata tgacc 25

<210> 15

<211> 9

<212> DNA

<213> Porcine circovirus type 3

<400> 15

tagtattac 9

<210> 16

<211> 19

<212> DNA

<213> Artificial sequence

<220>

<223> Internal cap gene primer

<400> 16

ccacagaagg cgctatgtc 19

<210> 17

<211> 19

<212> DNA

<213> Artificial sequence

<220>

<223> Internal cap gene primer

<400> 17

ccgcataagg gtcgtcttg 19

<210> 18

<211> 30

<212> DNA

<213> Artificial sequence

<220>

<223> Primer

<400> 18

aaaaaagcta gcgctggaac atactacaca 30

<210> 19

<211> 33

<212> DNA

<213> Artificial sequence

<220>

<223> Primer

<400> 19

aaaaaagaat tcttagagaa cggacttgta acg 33

<210> 20

<211> 5

<212> protein

<213> Artificial sequence

<220>

<223> Rolling circle replication motif

<400> 20

Phe Thr Leu Asn Asn

15

<210> 21

<211> 5

<212> protein

<213> Artificial sequence

<220>

<223> Rolling circle replication motif

<400> 21

Phe Thr Ile Asn Asn

15

<210> 22

<211> 4

<212> protein

<213> Artificial sequence

<220>

<223> Rolling ring replication motif

<400> 22

His Leu Gln Gly

1

<210> 23

<211> 4

<212> protein

<213> Artificial sequence

<220>

<223> Rolling ring replication motif

<400> 23

Tyr Cys Lys Lys

1

<210> 24

<211> 5

<212> protein

<213> Artificial sequence

<220>

<223> Motif of unknown origin

<400> 24

Trp Trp Asp Gly Tyr

15

<210> 25

<211> 8

<212> protein

<213> Artificial sequence

<220>

<223> Motif of unknown origin

<400> 25

Asp Asp Phe Tyr Gly Trp Val Pro

15

<210> 26

<211> 4

<212> protein

<213> Artificial sequence

<220>

<223> Motif of unknown origin

<400> 26

Asp Arg Tyr Pro

1

<210> 27

<211> 3

<212> DNA

<213> Artificial sequence

<220>

<223> Canonical start codon

<400> 27

gtc 3

<210> 28

<211> 3

<212> DNA

<213> Artificial sequence

<220>

<223> start codon

<400> 28

atg 3

<---

Claims (53)

1. A method of inducing an immunological response against porcine circovirus type 3, comprising the steps of: administering to an animal a composition containing a protein, wherein the protein contains an amino acid sequence having at least 90% sequence identity with SEQ ID NO. 6. 2. The method of claim 1, wherein the protein comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO. 6. 3. The method of claim 1, wherein the protein has been expressed in the insect cell from a baculovirus vector encoding the protein. 4. The method of claim 1, wherein the immunological response includes the production or activation of antibodies. 5. The method of claim 1, wherein said composition further comprises an additional component selected from the group consisting of an adjuvant, a pharmaceutically acceptable salt, an inactivating agent, a neutralizing agent, a portion of a cell culture supernatant, a veterinarily acceptable carrier, at least one immunologically active component against another pathogenic organism in pigs and any combination thereof. 6. The method of claim 5, wherein said adjuvant is selected from the group consisting of aluminum hydroxide, aluminum phosphate, saponin, emulsions, polymers of acrylic or methacrylic acid, copolymers of maleic anhydride and an alkenyl derivative, RIBI adjuvant system, block copolymer, SAF -M, monophosphoryl lipid A, avridine lipid amine, heat labile enterotoxin from E. coli, cholera toxin, IMS 1314 and muramyl dipeptide. 7. The method of claim 6, wherein said adjuvant is a carbomer. 8. The method of claim 7, wherein said carbomer is Carbopol or Carbopol 971P. 9. The method according to claim 6, wherein said adjuvant is an emulsion. 10. The method of claim 9, wherein said emulsion is a water-in-oil-in-water emulsion. 11. The method according to claim 10, wherein said emulsion contains mannide esters. 12. The method of claim 11, wherein said mannide esters comprise anhydromannite oleate. 13. The method of claim 5, wherein said inactivating agent is binary ethyleneimine (BEI). 14. The method of claim 5, wherein the veterinarily acceptable carrier is selected from the group consisting of a solvent, dispersion medium, coating, stabilizer, diluent, preservative, antimicrobial agent, antifungal agent, isotonic agent, adsorption retardant, or any combination thereof. 15. The method of claim 5, wherein said pharmaceutically acceptable salt is a phosphate salt in physiologically acceptable concentrations. 16. The method of claim 5, wherein said neutralizing agent is sodium thiosulfate. 17. A method of inducing an immunological response against porcine circovirus type 3, comprising the steps of: administering to an animal a composition containing a protein, where the protein is encoded by a nucleotide sequence having at least 90% sequence identity with SEQ ID NO. 5. 18. The method of claim 17, wherein the nucleotide sequence has at least 95% sequence identity to SEQ ID NO. 5. 19. The method of claim 17, wherein the protein has been expressed in the insect cell from a baculovirus vector encoding the protein. 20. The method of claim 17, wherein the immunological response includes the production or activation of antibodies. 21. The method of claim 17, wherein said composition further comprises an additional component selected from the group consisting of an adjuvant, a pharmaceutically acceptable salt, an inactivating agent, a neutralizing agent, a portion of a cell culture supernatant, a veterinarily acceptable carrier, at least one immunologically active component against another pathogenic organism in pigs and any combination thereof. 22. The method of claim 21, wherein said adjuvant is selected from the group consisting of aluminum hydroxide, aluminum phosphate, saponin, emulsions, polymers of acrylic or methacrylic acid, copolymers of maleic anhydride and an alkenyl derivative, RIBI adjuvant system, block copolymer, SAF -M, monophosphoryl lipid A, avridine lipid amine, heat labile enterotoxin from E. coli, cholera toxin, IMS 1314 and muramyl dipeptide. 23. The method of claim 22, wherein said adjuvant is a carbomer. 24. The method of claim 23, wherein said carbomer is Carbopol or Carbopol 971P. 25. The method according to claim 22, wherein said adjuvant is an emulsion. 26. The method of claim 25, wherein said emulsion is a water-in-oil-in-water emulsion. 27. The method according to claim 26, wherein said emulsion contains mannide esters. 28. The method of claim 27, wherein said mannide esters comprise anhydromannite oleate. 29. The method of claim 21, wherein said inactivating agent is binary ethyleneimine (BEI). 30. The method of claim 21, wherein the veterinary-acceptable carrier is selected from the group consisting of a solvent, dispersion medium, coating, stabilizer, diluent, preservative, antimicrobial agent, antifungal agent, isotonic agent, adsorption retardant, or any combination thereof. 31. The method of claim 21, wherein said pharmaceutically acceptable salt is a phosphate salt in physiologically acceptable concentrations. 32. The method of claim 21, wherein said neutralizing agent is sodium thiosulfate. 33. A method of inducing an immunological response against porcine circovirus type 3, comprising the steps of: administering to an animal a composition containing a vector containing a heterologous nucleotide sequence encoding a protein, where the protein contains an amino acid sequence having at least 90% sequence identity with SEQ ID NO. 6. 34. The method of claim 33, wherein the protein comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO. 6. 35. The method of claim 33, wherein the vector is a viral vector. 36. The method of claim 33, wherein the immunological response includes the production or activation of antibodies. 37. The method of claim 33, wherein said composition further comprises an additional component selected from the group consisting of an adjuvant, a pharmaceutically acceptable salt, an inactivating agent, a neutralizing agent, a portion of a cell culture supernatant, a veterinarily acceptable carrier, at least one immunologically active component against another pathogenic organism in pigs and any combination thereof. 38. The method of claim 37, wherein the veterinarily acceptable carrier is selected from the group consisting of a solvent, dispersion medium, coating, stabilizer, diluent, preservative, antimicrobial agent, antifungal agent, isotonic agent, adsorption retardant, or any combination thereof. 39. The method of claim 37, wherein said pharmaceutically acceptable salt is a phosphate salt in physiologically acceptable concentrations. 40. A method of inducing an immunological response against porcine circovirus type 3, comprising the steps of: administering to an animal a composition containing a vector containing a heterologous nucleotide sequence having at least 90% sequence identity with SEQ ID NO. 5. 41. The method of claim 40, wherein the nucleotide sequence has at least 95% sequence identity to SEQ ID NO. 5. 42. The method of claim 40, wherein the vector is a viral vector. 43. The method of claim 40, wherein the immunological response includes the production or activation of antibodies. 44. The method of claim 40, wherein said composition further comprises an additional component selected from the group consisting of an adjuvant, a pharmaceutically acceptable salt, an inactivating agent, a neutralizing agent, a portion of a cell culture supernatant, a veterinarily acceptable carrier, at least one immunologically active component against another pathogenic organism in pigs and any combination thereof. 45. The method of claim 44, wherein the veterinarily acceptable carrier is selected from the group consisting of a solvent, dispersion medium, coating, stabilizer, diluent, preservative, antimicrobial agent, antifungal agent, isotonic agent, adsorption retardant, or any combination thereof. 46. The method of claim 44, wherein said pharmaceutically acceptable salt is a phosphate salt in physiologically acceptable concentrations. 47. A method of inducing an immunological response against porcine circovirus type 3, comprising the steps of: administering to an animal a composition containing a vector containing a heterologous RNA sequence corresponding to a nucleotide sequence having at least 90% sequence identity with SEQ ID NO. 5. 48. The method of claim 47, wherein the nucleotide sequence has at least 95% sequence identity to SEQ ID NO. 5. 49. The method of claim 47, wherein the vector is a viral vector. 50. The method of claim 47, wherein the immunological response includes the production or activation of antibodies. 51. The method of claim 47, wherein said composition further comprises an additional component selected from the group consisting of an adjuvant, a pharmaceutically acceptable salt, an inactivating agent, a neutralizing agent, a portion of a cell culture supernatant, a veterinarily acceptable carrier, at least one immunologically active component against another pathogenic organism in pigs and any combination thereof. 52. The method of claim 51, wherein the veterinarily acceptable carrier is selected from the group consisting of a solvent, dispersion medium, coating, stabilizer, diluent, preservative, antimicrobial agent, antifungal agent, isotonic agent, adsorption retardant, or any combination thereof. 53. The method of claim 51, wherein said pharmaceutically acceptable salt is a phosphate salt in physiologically acceptable concentrations.