AU2006202480A1 - Porcine circo viruses, vaccines and diagnostic reagents I - Google Patents

Description

AUSTRALIA

Patents Act 1990 MERIAL; THE QUEEN'S UNIVERSITY OF BELFAST; UNIVERSITY OF SASKATCHEWAN COMPLETE SPECIFICATION STANDARD PATENT Invention Title.

Porcine circo viruses, vaccines and diagnostic reagents I The following statement is a full description of this invention including the best method of performing it known to us:- -Ic- 0 PORCINE CIRCOVIRUSES, VACCINES AND DIAGNOSTIC REAGENTS SThe present invention relates to new porcine circovirus (PCV for Porcine CircoVirus) strains responsible for the PMWS syndrome (Porcine Multisystemic Wasting Syndrome also called Post-Weaning Multisystemic Wasting Syndrome) to reagents and methods allowing their detection, to methods of vaccination and to vaccines, as well as to methods of producing these reagents and vaccines.

PCV was originally detected as a noncytopathogenic contaminant in pig kidney o1 cell lines PK/15. This virus was classified among the Circoviridae with the chicken anaemia virus (CAV for Chicken Anaemia Virus) and the PBFDV virus (Pscittacine Beak and Feather Disease Virus). It is a small nonenveloped virus (from 15 to 24 nm) whose common characteristic is that its genome is in the form of a circular single-stranded DNA of 1.76 to 2.31 kb. It was first thought that this genome encoded a polypeptide of about 30 kDa (Todd et al., Arch Virol 1991, 117; 129- 135); however, recent work has shown that it is more complex (Meehan B. M. et al., 1997, 78; 221-227). Moreover, there are no significant homologies in nucleotide sequence or in common antigenic determinants between the three types of known circoviruses.

The PCV derived from the PK/15 cells, PCV1, is considered not to be pathogenic.

Its sequence is known from B. M. Meehan et al., J. Gen. Virol 1997 (78) 221-227.

It is only very recently that some authors have thought that strains of PCV could be pathogenic and associated with the PMWS syndrome (Gupi P. S. Nayar et al., Can. Vet. J, vol. 38, 1997: 385-387 and Clark E. Proc. Am. Assoc. Swine Prac.

1997; 499-501). Nayar et al. have detected PCV DNA in pigs having the PMWS syndrome using PCR techniques. No wild-type PCV strain has however been isolated and purified so far.

The PMWS syndrome detected in Canada, the United States and France is clinically characterized by a gradual loss of weight and by manifestations such as tachypnea, dyspnea and jaundice. From the pathological point of view, it is manifested by lymphocytic or granulomateus infiltrations, lymphadenopathies and, P:%OPERjfna\Specirications%764379-200 120 DIV.doc- 2519/03 Smore rarely, by hepatitis and lymphocytic or granulomateus nephritis (Clark E. G., Proc. Am. Assoc. Swine Prac. 1997; 499-501; La Semaine Veterinaire No. 26, supplement to La Semaine Veterinaire 1996 (834); La Semaine Veterinaire 1997 (857): 54; Gupi P. S. Nayar et al., Can. Vet. J, vol. 38, 1997; 385-387).

00 The applicant has succeeded in isolating new PCV strains from pulmonary or CN ganglionic samples obtained from farms situated in Canada, the United States c (California) and France (Brittany), hereinafter called circoviruses according to the Sinvention. These viruses have been detected in lesions in pigs with the PMWS syndrome, but not in healthy pigs.

The applicant has, in addition, sequenced the genome of four of several strains. The strains exhibit a very strong homology with each other at the nucleotide level, exceeding 96% and much weaker homology with the PK/15 strain, about 76%. The new strains can thus be considered as being representative of a new type of porcine circovirus, called here type II (PCVII or PCV2), type I (PCVI or PCVI) being represented by The subject of the present invention is therefore the group II porcine circovirus, as defined above, isolated or in the form of a purified preparation.

According to one aspect, the present invention provides an isolated polynucleotide sequence coding for the PCVII specific amino acid sequence of Figure 10A or SEQ ID NO: 9.

In another aspect, the present invention provides an isolated polynucleotide sequence coding for the PCVII specific amino acid sequence of Figure In a further aspect, the present invention provides an isolated polynucleotide sequence coding for the PCVII specific amino acid sequence of Figure IOC or SEQ ID NO 13.

In yet a further aspect, the present invention provides an isolated polynucleotide sequence coding for the PCVII specific amino acid sequence of Figure 10D or SEQ ID NO m:\specifications\500000\502OX5022O9cImMjC.doc In another aspect, the present invention provides an isolated polynucleotide sequence 0 comprising the nucleotides 51 to 992 of Figure 11A.

In a further aspect, the present invention provides an isolated polynucleotide sequence oo 0 comprising a nucleotide sequence having at least 90% identity with the nucleotide Ssequence 51 to 992 of Figure 11A.

SIn a further aspect, the present invention provides an isolated polynucleotide sequence CN 10 comprising the nucleotides 671 to 360 or 565 to 389 of Figure 11 A.

In another aspect, the present invention provides an isolated polynucleotide sequence comprising a nucleotide sequence having at least 85% identity with the nucleotide sequence 671 to 360 or 565 to 389 of Figure 1 IA.

In a further aspect, the present invention provides an isolated polynucleotide sequence comprising a nucleotide sequence coding for 25 or more PCVII specific contiguous amino acids of Figure 10A or SEQ ID NO 9.

In another aspect, the present invention provides an isolated polynucleotide sequence comprising a nucleotide sequence coding for 25 or more PCVII specific contiguous amino acids of Figure In yet a further aspect, the present invention provides an isolated polynucleotide sequence comprising a nucleotide sequence coding for 25 or more PCVII specific contiguous amino acids of Figure 10C or SEQ ID NO 13.

In another further aspect, the present invention provides an isolated polynucleotide sequence comprising a nucleotide sequence coding for 25 or more PCVII specific contiguous amino acids of Figure 10D or SEQ ID NO It also provides an isolated polynucleotide sequence coding for 25 or more contiguous amino acids of a PCVII amino acid sequence having an homology of 80-85 or more with the PCVII amino acid sequence of Figure m:\specifications\500000\502000\502209cimmjc.doc In yet a further aspect, the present invention provides an isolated polypeptide t comprising the PCVII specific amino acid sequence of Figure 10A or SEQ ID NO: 9.

0 In another aspect, the present invention provides an isolated polypeptide comprising the PCVII specific amino acid sequence of Figure SIn a further aspect, the present invention provides an isolated polypeptide comprising the PCVII specific amino acid sequence of Figure O1C or SEQ ID NO 13.

In another aspect, the present invention provides an isolated polypeptide comprising the C 10 PCVII specific amino acid sequence of Figure 10D or SEQ ID NO In yet a further aspect, the present invention provides an isolated polypeptide comprising 25 or more PCVII specific contiguous amino acids of Figure 10A or SEQ ID NO: 9.

In a further aspect, the present invention provides an isolated polypeptide comprising or more PCVII specific contiguous amino acids of Figure In yet another further aspect, the present invention provides an isolated polypeptide comprising 25 or more PCVII specific contiguous amino acids of Figure 10C or SEQ ID NO: 13.

In another further aspect, the present invention provides an isolated polypeptide comprising 25 or more PCVII specific contiguous amino acids of Figure 10D or SEQ ID NO: In a further aspect, the present invention provides an isolated polypeptide comprising or more contiguous amino acids of a PCVII amino acid sequence having an homology of 80-85 or more with the PCVII amino acid sequence of Figure In another further aspect, the present invention provides an isolated PCVII nucleotide sequence of Figure 9A-9C or 10A-IOB, or SEQ ID NO 7,17,18 The invention relates to a porcine circovirus, particularly type II porcine circovirus, capable of being isolated from a physiological sample or from a tissue sample, m:\specifications5OOOOOl5O2OOO\5022O9clmmjc~doc especially lesions, from a diseased pig having PMWS, using the method described in the examples.

The subject of the present invention is purified preparations of five strains, which were deposited at the ECACC (European Collection of Cell Cultures, Centre for Applied oO Microbiology Research, Porton Down, Salisbury, Wiltshire SP4 OJG, United Kingdom) on Thursday Oct. 2, 1997: S- provisional accession No. V97100219 (called here Imp. 1008PCV) provisional accession No. V97100218 (called here Imp. 1010PCV) 1 10 provisional accession No. V97100217 (called here Imp. 999PCV), and, m:\specificaions\500000\502000\52209clmmjc.doc on Friday Jan. 16 1998 provisional accession No. V98011608 (called here Imp. 1011-48285) provisional accession No. V98011609 (called here Imp. 1011-48121).

O

The invention describes further isolation of PCVII 412, as well as several Sadditional PCVII isolates.

oo SThe invention aims to consider the porcine circoviruses isolated from a diseased Spig and/or the circoviruses having a significant serological similarity with the o0 strains of the invention and/or the circoviruses having cross-hybridization with the strains of the invention under stringency conditions such that there is no hybridization with the PCV PK/15 strain.

The viral strains isolated from a physiological sample or from a tissue sample, especially a lesion, from a pig having the PMWS syndrome can be advantageously propagated on cell lines such as pig kidney cell lines, for example, cells free from PCV contamination. Such propagation is useful for producing the whole virus or for the production of antigens, such as polypeptide subunits.

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

The subject of the present invention is also the preparations of circoviruses isolated after passages on cells, especially cell lines, e.g. PK/15 cells, cultured in vitro while being infected with at least one of the circoviruses according to the invention or of any porcine circovirus capable of being isolated form a physiological sample or from a tissue sample, especially lesions, from a pig having the PMWS syndrome. Its subject is also the culture extract or supernatant, optionally purified by standard techniques, and in general any antigenic preparation obtained from in vitro cultures.

P:\OPER\jn\Specifications\764379-2002302120 DIV.doc- 25/9/03 SThe subject of the invention is also the immunogenic active ingredients and the vaccines containing at least one antigen as defined above.

They may be immunogenic active ingredients based on attenuated live whole Sviruses, or vaccines prepared with these active ingredients, the attenuation being Scarried out according to the customary methods, e.g. by passage on cells, Spreferably by passage on pig cells, especially lines, such as PK/15 cells (for Sexample from 50 to 150, preferably on the order of 100, passages). These io vaccines can comprise a veterinarilly acceptable vehicle, diluent, adjuvant, and/or a freeze-drying stabilizer.

These vaccines will preferably comprise from 10 3 to 10 6 They may be immunogenic active ingredients or vaccines based on circovirus antigen according to the invention, in an inactivated state. The vaccine comprises, in addition, a veterninarilly acceptable vehicle or diluent, and optionally, an adjuvant.

The circoviruses according to the invention, with the fractions which may be present, are inactivated according to techniques known to persons skilled in the art. The inactivation will be preferably carried out by the chemical route, e.g. by exposing the antigen to a chemical agent such as formaldehyde (formalin), paraformaldehyde, 0-propiolactone or ethyleneimine or its derivatives. The preferred method of inactivation will be herein the exposure to a chemical agent and in particular to ethyleneimine or to P-propiolactone.

Preferably, the inactivated vaccines according to the invention will be supplemented with adjuvant, advantageously by being provided in the form of emulsions, for example water-in-oil or oil-in-water, according to techniques well known to persons skilled in the art. It will be possible for the adjuvant character to also come from the addition of a customary adjuvant compound to the active ingredient.

P:%OPERljna.Spcciicaioni76A379-.2Q20 2 I 20 DIVdoc- 259103

INC

SAmong the adjuvants which may be used, there may be mentioned by way of example aluminium hydroxide, the saponines Quillaja saponin or Quil A; see Vaccine Design, The Subunit and Adjuvant Approach, 1995, edited by Michael F.

Powel and Mark J. Newman, Plennum Press, New-York and London, p.210), SAvridine.RTM. (Vaccine Design p. 148), DDA (Dimethyldioctadecylammonium Sbromide, Vaccine Design p. 157), Polyphosphazene (Vaccine Design p. 204), or alternatively oil-in-water emulsions based on mineral oil, squalane SPT Semulsion, Vaccine Design p. 147), squalene MF59, Vaccine Design p. 183), or water-in-oil emulsions based on metabolizable oil (preferably according to WO- A-94 20071) as well as the emulsions described in U.S. Pat. No. 5,422,109. It is also possible to choose combinations of adjuvants, for example Avridine.RTM. or DDA combined with an emulsion.

These vaccines will preferably comprise from 106 to 108 The live vaccine adjuvants can be selected from those given for the inactivated vaccine. Emulsions are preferred; some examples are described in WO-A- 9416681.

Several compounds can be used as a freeze-drying stabilizer, for example SPGA (Bovarnik et al., J. Bacteriology 59, 509, 950), carbohydrates such as sorbitol, mannitol, starch, sucrose, dextran or glucose, proteins such as albumin or casein, derivatives of these compounds, or buffers such as alkali metal phosphates.

The entire DNA genomes of seven isolates of this novel PCV have been cloned and sequenced. Portions of these DNA sequences are useful as probes to diagnose the presence of virus in clinical samples, and to isolate other naturally occurring variants of the virus. An understanding of the genomic sequence of PCVII also makes available the polypeptide sequences of the various proteins encoded within the open reading frames of the viral genome and permits production of these peptides or portions thereof which are useful as standards or reagents in diagnostic tests and as components of vaccines. Protective antibodies P:%OPER~jn\Speciricwions%764379-200202120 DIV.doc 2/9/03 O may also be raised from the proteins and may be produced in polyclonal or Smonoclonal form.

The availability of the entire PCVII sequence thus permits the design and construction of polypeptides which may either serve as vaccines or diagnostic 0 reagents, or as intermediates in the production of monoclonal antibody (Mab) t preparations useful in passive immunotherapy against PMWS, or as intermediates 0 in the production of antibodies useful as diagnostic reagents.

ON

to The subject of the present invention is therefore a DNA fragment containing all or part of one of these sequences. It goes without saying that the invention automatically covers the equivalent sequences, that is to say the sequences which do not change the functionality or the strain-specificity of the sequence described or of the polypeptides encoded by this sequence. There will of course be included the sequences differing by degeneracy of the genetic code.

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

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

In particular, the open reading frames, forming DNA fragments according to the invention, which can be used to this effect have been identified on the genomic sequence of the type II circoviruses. The invention relates to any polypeptide containing at least one of these open reading frames (corresponding amino acid sequence).

Accordingly, in one aspect, the invention relates to polynucleotides useful for the production of PCVII diagnostics and vaccines derived from the PCVII genome. In one particular embodiment, the polynucleotides are capable of selectively PA OPEJrmSpcir:Iorns76439.-20232 120 DIV.dx 23/91O 0 hybridizing to a PCVII nucleotide sequence and comprise at least about 8 Scontiguous nucleotides derived from, or complementary to, a PCVII sequence depicted in FIGS. 11 (SEQ ID NO:7, SEQ ID NO:17 and SEQ ID NO:18 30). In another embodiment, the polynucleotide encodes an immunogenic PCVII polypeptide having at least about 85% identity to a polypeptide selected from the Sgroup consisting of a polypeptide derived from ORF 1 (SEQ ID NO:9), ORF 2 (SEQ ID NO:11), ORF 3 (SEQ ID NO:15), ORF 4 (SEQ ID NO:13), (e) SORF 5 (SEQ ID NO:26), ORF 6 (SEQ ID NO:27), and immunogenic N fragments of comprising at least about 5 amino acids. In a particularly to preferred embodiment, the polynucleotide encodes the polypeptide of ORF 2 (SEQ ID NO:11), or immunogenic fragments thereof.

The invention thus relates to utilizing these polynucleotide sequences or portions thereof as oligomeric probes, for production of peptides which can serve as diagnostic reagents or as vaccine antigens, to the peptides themselves, and to polyclonal and monoclonal antibodies useful in diagnosis and treatment of the disease.

Other aspects of the invention include expression systems which are capable of effecting the production of a desired protein encoded by sequences derived from the complete genome, recombinant vectors containing such systems or portions thereof, recombinant host cells transformed with such vectors, proteins produced by the transformed cells, and vaccines prepared from such proteins. In addition, the invention relates to peptide sequences representing epitopes encoded by the genome, and to such sequences covalently linked to label or to carrier proteins.

Also encompassed by the present invention are the various ORFs of the PCVII genome, as well as the proteins encoded by these ORFs, and portions thereof.

Preferably, the invention relates to a protein essentially consisting of ORF1, ORF2, ORF3 or ORF4.

For the expression of subunits in vitro, as a means of expression, E. coli or a baculovirus will be preferably used Pat. No. 4,745,051). The coding P:IOPERljlm\Spciricaiions%764379-2003 120 DIV.doc.- 2519M0 O sequence(s) or their fragments are integrated into the baculovirus genome (e.g.

Sthe baculovirus Autographa californica Nuclear Polyhedrosis Virus AcNPV) and the latter is then propagated on insect cells, e.g. Spodoptera frugiperda Sf9 (deposit ATCC CRL 1711). The subunits can also be produced in eukaryotic cells such as yeasts Saccharomyces cerevisiae) or mammalian cells CHO, O BHK).

00 SThe subject of the invention is also the polypeptides which will be produced in vitro °by these expression means, and then optionally purified according to conventional techniques. Its subject is also a subunit vaccine comprising at least one polypeptide as thus obtained, or fragment, in a veterinarilly acceptable vehicle or diluent and optionally a veterinarilly acceptable adjuvant.

For the expression in vivo for the purpose of producing recombinant live vaccines, the coding sequence(s) or their fragments are inserted into an appropriate expression vector under conditions allowing the expression of the polypeptide(s).

As appropriate vectors, there may be used live viruses, preferably capable of multiplying in pigs, nonpathogenic for pigs (naturally nonpathogenic or rendered as such), according to techniques well known to persons skilled in the art. There may be used in particular pig herpesviruses such as Aujeszky's disease virus, porcine adenovirus, poxviruses, especially vaccinia virus, avipox virus, canarypox virus, swinepox virus. Plasmid DNAs can also be used as vectors (WO-A-90 11092, WO-A-93 19813, WO-A-94 21797, WO-A-95 20660).

The subject of the invention is therefore also the vectors and the recombinant live vaccines or plasmid vaccines (polynucleotide or DNA vaccines) thus prepared.

The vaccines can additionally comprise a veterinarilly acceptable vehicle or diluent.

The vaccine according to the invention (live attenuated, inactivated, sub-units, recombinant and plasmid vaccines) may comprise one or more active ingredients (antigens) of one or more (2 or 3) of the circoviruses according to the invention.

P:AOPERjm Specirniaions%764379.2002302 120 DIV.doc. 25M9103 SFor each vaccine type as described above, the invention also provides for 0 combining vaccination against the porcine circovirus with a vaccination against other pig pathogens, in particular those which can be associated with the PMWS O syndrome. The vaccines according to the invention, in particular the inactivated ones, may therefore comprise another valency corresponding to another pig oo pathogen. Other porcine pathogens include PRRS (Porcine Reproductory and SRespiratory Syndrome) (the one skilled in the art may refer to WO-A-93/07898, SWO-A-94/18311, FR-A-2 709 966 C. Chareyre et al., Proceedings of the 15 th

ID

0 IPVS Congress, Birmingham, England, 5-9 July 1998, p 139; incorporated herein c 10 by reference), Mycoplasma hyopneumoniae (the one skilled in the art may refer to EP-A-597 852, EP-A-550, 477, EP-A-571 648 0. Martinon et al. p 157, 284, 285 and G. Reynaud et al., p 150, all in the above-referenced Proceedings of the 15 th IPVS Congress incorporated herein by reference), Actinobacillus pleuropneumoniae, E. coli. Porcine Atrophic Rhinitis, Pseudorabies (Aujeszky disease), Hog cholera, and Swine Influenza.

The subject of the present invention is also a method which makes it possible to induce an immune response in pigs towards circoviruses according to the invention. Also contemplated is a method of vaccination which is effective in pigs.

This method provides for the administration to pigs, in once or several times, of a vaccine above. It is also possible to combine several types of the above vaccines in the same vaccination protocol.

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

The invention also offers the possibility of diagnosing the presence of the circoviruses according to the invention in pigs. One embodiment is therefore diagnostic tests and methods relating thereto using reagents which will be described below.

P: OPERjnr,icationsV763792002302 120 DIV.doc- 2519103 SKnowledge of the sequences of the different circoviruses makes it possible to define common sequences which make it possible to produce reagents capable of o recognizing all the porcine circoviruses known.

SPersons skilled in the art will also be able to select fragments of the sequences 00 corresponding to regions exhibiting little or no homology with the corresponding 0 PK/15 circovirus sequence in order to carry out a specific diagnosis.

NO

Sequence alignments make it possible for persons skilled in the art to select a reagent in accordance with their desired utility.

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

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

A third and fourth reagent consists in respectively polyclonal and monoclonal antibodies which may be produced according to the customary techniques from the virus, the polypeptides or fragments, extracted or encoded by the DNA sequences.

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

P:%OPERj,1\Spcirlcohu1is643792002302 120 DIVdoc- 2519/03 SThe antigens and antibodies according to the invention may be used in any known laboratory diagnostic technique.

However, it will be preferable to use them in techniques which can be used directly 0 in the field by the veterinary doctor, the breeder or the owner of the animal.

o00 Persons skilled in the art have available a range of laboratory and field techniques Sand are therefore in the perfect position to adapt the use of this antigen and/or N antibodies as diagnostic reagent(s).

The diagnostic techniques which will be preferably used within the framework of the present invention are Western blotting, immunofluoroescence, ELISA and immunochromatography.

As regards the use of immunochromatography methods, specialists can refer in particular to Robert F. Zurk et al., Clin. Chem. 31/7, 1144-1150 (1985) as well as to patents or patent applications WO-A-88/08 534, WO-A-91/12528, EP-A-291 176, EP-A-299 428, EP-A-291 194, EP-A-284 232, U.S. Pat. No. 5,120,643, U.S.

Pat. No. 5,030,558, U.S. Pat. No. 5,266,497, U.S. Pat. No. 4,740,468, U.S. Pat.

No. 5,266,497, U.S. Pat. No. 4,855,240, U.S. Pat. No. 5,451,504, U.S. Pat. No.

5,141,850, U.S. Pat. No. 5,232,835 and U.S. Pat. No. 5,238,652.

Accordingly, it is preferably sought to detect specific antibodies in the sample by an indirect test, by competition or by displacement. To do this, the antigen itself is used as diagnostic reagent, or a fragment of this antigen, conserving recognition of the antibodies. The labelling may be advantageously a labelling with peroxidase or a special labelling, for example, colloidal gold.

It may also be desired to detect the antigen itself in the sample with the aid of a labelled antibody specific for this antigen. The labelling is advantageously as described above.

By antibody specific for the antigen which can be used in particular in competition P:\OPER~jrmsSpccificaiions'76d792002302 120 DIV.doc. 25IO3 O or displacement or for the detection of the antigen itself, there is understood Smonoclonal or polyclonal antibodies specific for the antigen, fragments of these antibodies, for example, Fab or F(ab) 2 fragments.

Another feature of the invention is the production of polyclonal or monoclonal Santibodies specific for the antigen in accordance with the invention.These antibodies can be used as diagnostic reagent for the detection of the antigen in a Ssample of physiological fluid or in a tissue sample, or even for the detection of Santibodies present in such a sample or specimen. The invention also includes the io immunologically functional fragments of these antibodies, the F(ab) and F(ab) 2 fragments.

Antibodies can be prepared by the customary techniques. Reference may be made in particular to Antibodies, A Laboratory Manual, 1988, Cold Spring Harbor Laboratory, USA or to J. W. Goding, Monoclonal Antibodies: Principles and Practice, Academic Press Inc., whose contents are incorporated herein by reference.

Standard, known procedures can be used to carry out the fusion of spleen cells of mice, immunized with the antigen or with at least one of its fragments, with suitable myelomatous cells, to produce monoclonal antibodies.

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

The present invention also makes it possible to determine epitopes of interest especially on the basis of the DNA sequences described here, whether epitopes of vaccinal interest or epitopes of interest in diagnosis. From the DNA sequence of the genome of the circovirus according to the invention, persons skilled in the art are in a position to determine epitopes according to known methods, for example an appropriate computer program or PEPSCAN. Epitopes are immunodominant regions of proteins and are as such regions exposed at the surface of the proteins.

P:%OPER~jrra\SpeiriccJI ons\7637920023D2 0O DIV.doc 23/9/03 They can therefore be recognized by antibodies and thus be used in the field of N diagnosis either for the preparation of antibodies for diagnostic purposes or for the production of corresponding peptides which can be used as diagnostic reagents.

At the very least, an epitope is a peptide having from 8 to 9 amino acids. A O minimum of 13 to 25 amino acids is generally preferred.

oo O Persons skilled in the art are therefore in a position, using one or more of these N techniques as well as the other available techniques, to find epitopes for using O 10 peptides or antibodies for diagnostic purposes.

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

One object of the invention is a method of detecting PCVII antibodies in a biological sample comprising: providing a biological sample; reacting the biological sample with an immunogenic PCVII polypeptide as described above, under conditions which allow PCVII antibodies, when present in the biological sample, to bind to the PCVII polypeptide to form an antibody/antigen complex; and detecting the presence or absence of the complex, thereby detecting the presence or absence of PCVII antibodies in the sample.

P:OPER'jmsSpccitrscrnions764379.2O22 120 DIV.doc. 25f/03 SIn another embodiment, the invention is directed to a nucleic acid hybridization Sassay for detecting PCVII homologous sequences in a biological sample comprising: incubating the biological sample with a polynucleotide according to claim 1 under conditions which promote the formation of nucleic acid complexes Sbetween the polynucleotide and PCVII nucleic acid present in the biological Ssample; and N detecting the complexes containing the polynucleotide.

DETAILED DESCRIPTION The practice of the present invention will employ, unless otherwise indicated, conventional techniques of molecular biology, microbiology, recombinant DNA technology, and immunology, which are within the skill of the art. Such techniques are explained fully in the literature. See, Sambrook, Fritsch Maniatis, Molecular Cloning: A Laboratory Manual, Vols. I, II and III, Second Edition (1989); DNA Cloning, Vols. I and II N. Glover ed. 1985); Oligonucleotide Synthesis (M.

J. Gait ed. 1984); Nucleic Acid Hybridization D. Hames S. J. Higgins eds.

1984); Animal Cell Culture K. Freshney ed. 1986); Immobilized Cells and Enzymes (IRL press, 1986); Perbal, A Practical Guide to Molecular Cloning (1984); the series, Methods In Enzymology Colowick and N. Kaplan eds., Academic Press, Inc.); and Handbook of Experimental Immunology, Vols. I-IV (D.

M. Weir and C. C. Blackwell eds., 1986, Blackwell Scientific Publications). Before describing the present invention in detail, it is to be understood that this invention is not limited to particular DNA, polypeptide sequences or process parameters and, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments of the invention only, and is not intended to be limiting.

It must be noted that, as used in this specification and the appended claims, the singular forms "an" and "the" include plural referents unless the content clearly dictates otherwise. Thus, for example, reference to "an antigen" includes a mixture P:IOPER~jm,'SpecirTmonV764379.-2023O1 120 DIV.dc. 25/9/03 Q of two or more antigens, reference to "an excipient" includes mixtures of two or Smore excipients, and the like.

0 The following amino acid abbreviations are used throughout the text: Alanine: Ala Arginine: Arg Asparagine: Asn Aspartic acid: Asp (D) 0 Cysteine: Cys Glutamine: Gin Glutamic acid: Glu Glycine: Gly (G) Histidine: His Isoleucine: lie Leucine: Leu Lysine: Lys Methionine: SMet Phenylalanine: Phe Proline: Pro Serine: Ser Threonine: Thr (T) 0Tryptophan: Trp Tyrosine: Tyr Valine: Val (V) S A. Definitions Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention pertains. Although a number of methods and materials similar or equivalent to those described herein can be used in the practice of the present invention, the preferred materials and methods are described herein.

In describing the present invention, the following terms will be employed, and are intended to be defined as indicated below.

The terms "PCVII protein," "PMWS protein" or a nucleotide sequence encoding the same, intend a protein or a nucleotide sequence, respectively, which is derived from a novel PCVII isolate, as described herein. The nucleotide sequences of several PCVII isolates are shown in the Figures and the amino acid sequences corresponding to the several identified PCVII ORFs are also shown in the Figures.

However, a PCVII or PMWS protein, or a gene encoding the same, as defined herein is not limited to the depicted sequence.

Further, as used herein, a nucleotide sequence "derived from" a PCVII genome or its complement refers to a sequence which retains the essential properties of the illustrated polynucleotide, representing a portion of the entire sequence from which it is derived, for the purpose intended. A specific, but nonlimiting, example of such P:%OPER~jrsSpccificc oni764379.2023021 20 DIVdoc 21//03 O derivation is represented by a sequence which encodes an identical or Ssubstantially identical amino acid sequence, but, because of codon degeneracy, utilizes different specific codons; another example is a sequence complementary o to the viral DNA. A probe or oligonucleotide useful in diagnostic tests needs to retain the complementarity of the sequence shown but may be shorter than the 0 entire sequence or may skip over portions of it. However, for use in manipulation 00 or expression, nucleotide changes are often desirable to create or delete 0 restriction sites, provide processing sites, or to alter the encoded amino acid sequence in ways which do not adversely affect functionality. The terms "Nucleotide sequence" and "polynucleotide" refer both to ribonucleotide and a deoxyribonucleotide sequences and include both the genomic strand and its complementary sequence.

A sequence "derived from" the nucleotide sequence which comprises the genome of a PCVII isolate therefore refers to a sequence which is comprised of a sequence corresponding to a region of the genomic nucleotide sequence (or its complement), or a combination of regions of that sequence modified in ways known in the art to be consistent with its intended use. These sequences are, of course, not necessarily physically derived from the nucleotide sequence of the gene, but refer to polynucleotides generated in whatever manner which are based on the information provided by the sequence of bases in the region(s) from which the polynucleotide is derived. For example, regions from which typical DNA sequences can be "derived" include regions encoding specific epitopes. Similarly, a peptide "derived from" a PCVII ORF refers to an amino acid sequence substantially identical to that of these polypeptides or a portion thereof, having the same biological properties as that portion.

Furthermore, the derived protein or nucleotide sequences need not be physically derived from the genes described above, but may be generated in any manner, including for example, chemical synthesis, isolation from a PCVII isolate) or by recombinant production, based on the information provided herein. Additionally, the term intends proteins having amino acid sequences substantially homologous P \OPERj.rg pccirlcs:.ons'764379.2002302120 DIV.doc- 251Q103 O (as defined below) to contiguous amino acid sequences encoded by the genes, Swhich display immunological activity.

Thus, the terms intend full-length, as well as immunogenic, truncated and partial s sequences, and active analogs and precursor forms of the proteins. Also included 0 in the term are nucleotide fragments of the particular gene that include at least 00 Sabout 8 contiguous base pairs, more preferably at least about 10-20 contiguous 0 base pairs, and even at least about 25 to 50 or 75 or more contiguous base pairs N of the gene. Such fragments are useful as probes, in diagnostic methods, and for Sto the recombinant production of proteins, as discussed more fully below.

The terms also include proteins in neutral form or in the form of basic or acid addition salts depending on the mode of preparation. Such acid addition salts may involve free amino groups and basic salts may be formed with free carboxyls.

Pharmaceutically acceptable basic and acid addition salts are discussed further below. In addition, the proteins may be modified by combination with other biological materials such as lipids and saccharides, or by side chain modification, such as acetylation of amino groups, phosphorylation of hydroxyl side chains, oxidation of sulfhydryl groups, glycosylation of amino acid residues, as well as other modifications of the encoded primary sequence.

The term therefore intends deletions, additions and substitutions to the sequence, so long as the polypeptide functions to produce an immunological response as defined herein. In this regard, particularly preferred substitutions will generally be conservative in nature, those substitutions that take place within a family of amino acids. For example, amino acids are generally divided into four families: (1) acidic--aspartate and glutamate; basic--lysine, arginine, histidine; non-polar- -alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan; and uncharged polar--glycine, asparagine, glutamine, cystine, serine threonine, tyrosine. Phenylalanine, tryptophan, and tyrosine are sometimes classified as aromatic amino acids. For example, it is reasonably predictable that an isolated replacement of leucine with isoleucine or valine, or vice versa; an aspartate with a glutamate or vice versa; a threonine with a serine or vice versa; or P:\OPER\jns Specilrcations\764379-.002302120 DIV.doc 25/9103 O a similar conservative replacement of an amino acid with a structurally related Samino acid, will not have a major effect on the biological activity. Proteins having substantially the same amino acid sequence as the reference molecule, but possessing minor amino acid substitutions that do not substantially affect the immunogenicity of the protein, are therefore within the definition of the reference 0 polypeptide.

00 SAn "open reading frame" or "ORF" is a region of a polynucleotide sequence which N encodes a PCV polypeptide.

By "postweaning multisystemic wasting syndrome" or "PMWS" is meant a disease of vertebrate animals, in particular pigs, which is characterized clinically by progressive weight loss, tachypnea, dyspnea and jaundice. Consistent pathologic changes include lymphocytic to granulomatous interstitial pneumonia, lymphadenopathy, and, less frequently, lymphocytic to granulomatous hepatitis and nephritis. See, Clark, E. G. Proc. Am. Assoc. Swine Pract. 1997:499-501; and Harding, J. Proc. Am. Assoc. Swine Pract. 1997:503.

An "isolated" nucleic acid molecule is a nucleic acid molecule separate and discrete from the whole organism with which the molecule is found in nature; or a nucleic acid molecule devoid, in whole or part, of sequences normally associated with it in nature; or a sequence, as it exists in nature, but having heterologous sequences (as defined below) in association therewith.

The term "vaccine composition" intends any pharmaceutical composition containing an antigen, which composition can be used to prevent or treat a disease or condition in a subject. The term thus encompasses subunit vaccines, recombinant vaccines, as well as compositions containing whole killed, attenuated or inactivated virus.

By "subunit vaccine composition" is meant a composition containing at least one immunogenic polypeptide, but not all antigens, derived from or homologous to an antigen from a pathogen of interest. Such a composition is substantially free of P.AOPERjna\Spciriionans76437Q*02D2I1ZO DIV.doc 25/9/03 O intact pathogen cells or particles, or the lysate of such cells or particles. Thus, a S"subunit vaccine composition" is prepared from at least partially purified (preferably substantially purified) immunogenic polypeptides from the pathogen, or recombinant analogs thereof. A subunit vaccine composition can comprise the subunit antigen or antigens of interest substantially free of other antigens or Spolypeptides from the pathogen.

The term "epitope" refers to the site on an antigen or hapten to which specific B Icells and/or T cells respond. The term is also used interchangeably with "antigenic determinant" or "antigenic determinant site." Antibodies that recognize the same epitope can be identified in a simple immunoassay showing the ability of one antibody to block the binding of another antibody to a target antigen.

An "immunological response" to a composition or vaccine is the development in the host of a cellular and/or antibody-mediated immune response to the composition or vaccine of interest. Usually, an "immunological response" includes but is not limited to one or more of the following effects: the production of antibodies, B cells, helper T cells, suppressor-T cells, and/or cytotoxic T cells and/or .gamma..delta. T cells, directed specifically to an antigen or antigens included in the composition or vaccine of interest. Preferably, the host will display either a therapeutic or protective immunological response such that resistance to new infection will be enhanced and/or the clinical severity of the disease reduced.

Such protection will be demonstrated by either a reduction or lack of symptoms normally displayed by an infected host, a quicker recovery time and/or a lowered viral titer in the infected host.

The terms "immunogenic" protein or polypeptide refer to an amino acid sequence which elicits an immunological response as described above. An "immunogenic" protein or polypeptide, as used herein, includes the full-length sequence of the protein, analogs thereof, or immunogenic fragments thereof. By "immunogenic fragment" is meant a fragment of a protein which includes one or more epitopes and thus elicits the immunological response described above. Such fragments can be identified using any number of epitope mapping techniques, well known in the P;%OPERjrmSpecirictions764379-200202 120 DIV.doc. 25/9/03 O art. See, Epitope Mapping Protocols in Methods in Molecular Biology, Vol. 66 S(Glenn E. Morris, Ed., 1996) Humana Press, Totowa, N.J. For example, linear epitopes may be determined by concurrently synthesizing large numbers of peptides on solid supports, the peptides corresponding to portions of the protein molecule, and reacting the peptides with antibodies while the peptides are still 0 attached to the supports. Such techniques are known in the art and described in, U.S. Pat. No. 4,708,871; Geysen et al. (1984) Proc. Natl. Acad. Sci. USA S81:3998-4002; Geysen et al. (1986) Molec. Immunol. 23:709-715, all incorporated Sherein by reference in their entireties. Similarly, conformational epitopes are Sio readily identified by determining spatial conformation of amino acids such as by, x-ray crystallography and 2-dimensional nuclear magnetic resonance. See, Epitope Mapping Protocols, supra.

Synthetic antigens are also included within the definition, for example, polyepitopes, flanking epitopes, and other recombinant or synthetically derived antigens. See, 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, Jun. 28-Jul. 3, 1998.

Immunogenic fragments, for purposes of the present invention, will usually include at least about 3 amino acids, preferably at least about 5 amino acids, more preferably at least about 10-15 amino acids, and most preferably 25 or more amino acids, of the molecule. There is no critical upper limit to the length of the fragment, which could comprise nearly the full-length of the protein sequence, or even a fusion protein comprising two or more epitopes of the protein.

"Native" proteins or polypeptides refer to proteins or polypeptides isolated from the source in which the proteins naturally occur. "Recombinant" polypeptides refer to polypeptides produced by recombinant DNA techniques; produced from cells transformed by an exogenous DNA construct encoding the desired polypeptide.

"Synthetic" polypeptides are those prepared by chemical synthesis.

P;%OPEInmsiSpeciinctionsl764379202302 120 DIV.doc. 25/9103 SA DNA "coding sequence" -or a "nucleotide sequence encoding" a particular protein, is a DNA sequence which is transcribed and translated into a polypeptide in vitro or in vivo when placed under the control of appropriate regulatory elements. The boundaries of the coding sequence are determined by a start codon Sat the 5' (amino) terminus and a translation stop codon at the 3' (carboxy) terminus. A coding sequence can include, but is not limited to, procaryotic Ssequences, cDNA from eucaryotic mRNA, genomic DNA sequences from Seucaryotic mammalian) DNA, and even synthetic DNA sequences. A transcription termination sequence will usually be located 3' to the coding sequence.

DNA "control elements" refers collectively to promoters, ribosome binding sites, polyadenylation signals, transcription termination sequences, upstream regulatory domains, enhancers, and the like, which collectively provide for the transcription and translation of a coding sequence in a host cell. Not all of these control sequences need always be present in a recombinant vector so long as the desired gene is capable of being transcribed and translated.

"Operably linked" refers to an arrangement of elements wherein the components so described are configured so as to perform their usual function. Thus, control elements operably linked to a coding sequence are capable of effecting the expression of the coding sequence. The control elements need not be contiguous with the coding sequence, so long as they function to direct the expression thereof. Thus, for example, intervening untranslated yet transcribed sequences can be present between a promoter and the coding sequence and the promoter can still be considered "operably linked" to the coding sequence.

A control element, such-as a promoter, "directs the transcription" of a coding sequence in a cell when RNA polymerase will bind the promoter and transcribe the coding sequence into mRNA, which is then translated into the polypeptide encoded by the coding sequence.

PAOPERljmh.peikociakmns7643792002302 120 DIV.doc 25/9/03 SA "host cell" is a cell which has been transformed, or is capable of transformation, by an exogenous nucleic acid molecule.

A cell has been "transformed" by exogenous DNA when such exogenous DNA has been introduced inside the cell membrane. Exogenous DNA may or may not be integrated (covalently linked) into chromosomal DNA making up the genome of the cell. In procaryotes and yeasts, for example, the exogenous DNA may be Smaintained on an episomal element, such as a plasmid. With respect to eucaryotic cells, a stably transformed cell is one in which the exogenous DNA has become integrated into the chromosome so that it is inherited by daughter cells through chromosome replication. This stability is demonstrated by the ability of the eucaryotic cell to establish cell lines or clones comprised of a population of daughter cells containing the exogenous DNA.

"Homology" refers to the percent identity between two polynucleotide or two polypeptide moieties. Two DNA, or two polypeptide sequences are "substantially homologous" to each other when the sequences exhibit at least about 80%-85%, preferably at least about 90%, and most preferably at least about 95%-98% sequence identity over a defined length of the molecules. As used herein, substantially homologous also refers to sequences showing complete identity to the specified DNA or polypeptide sequence.

Percent identity can be determined by a direct comparison of the sequence information between two molecules by aligning the sequences, counting the exact number of matches between the two aligned sequences, dividing by the length of the shorter sequence, and multiplying the result by 100. Readily available computer programs can be used to aid in the analysis, such as ALIGN, Dayhoff, M. O. in Atlas of Protein Sequence and Structure M. O. Dayhoff ed., 5 Suppl.

3:353-358, National biomedical Research Foundation, Washington, which adapts the local homology algorithm of Smith and Waterman (1981) Advances in Appl. Math. 2:482-489 for peptide analysis. Programs for determining nucleotide sequence identity are available in the Wisconsin Sequence Analysis Package, P:\OPER\jrmsSpccincaikns\764379-2002302120 DIV.doc- 25/9/03 O Version 8 (available from Genetics Computer Group, Madison, Wis.) for example, Sthe BESTFIT, FASTA and GAP programs, which also rely on the Smith and Waterman algorithm. These programs are readily utilized with the default parameters recommended by the manufacturer and described in the Wisconsin Sequence Analysis Package referred to above.

00 Alternatively, homology can be determined by hybridization of polynucleotides 0 under conditions which form stable duplexes between homologous regions, followed by digestion with single-stranded-specific nuclease(s), and size S 10 determination of the digested fragments. DNA sequences that are substantially homologous can be identified in a Southern hybridization experiment under, for example, stringent conditions, as defined for that particular system. Defining appropriate hybridization conditions is within the skill of the art. See, e.g., Sambrook et al., supra; DNA Cloning, supra; Nucleic Acid Hybridization, supra.

Two nucleic acid fragments are considered to be "selectively hybridizable" to a PCVII polynucleotide, if they are capable of specifically hybridizing to a PCVII nucleic acid or a variant thereof a probe that hybridizes to a PCVII nucleic acid but not to polynucleotides from other members of the circovirus family) or specifically priming a polymerase chain reaction: under typical hybridization and wash conditions, as described, for example, in Sambrook et al., supra and Nucleic Acid Hybridization, supra, (ii) using reduced stringency wash conditions that allow at most about 25-30% basepair mismatches, for example: 2 x SSC, 0.1% SDS, room temperature twice, 30 minutes each; then 2 x SSC, 0.1% SDS, 37"C once, 30 minutes; then 2 x SSC room temperature twice, 10 minutes each, or (iii) selecting primers for use in typical polymerase chain reactions (PCR) under standard conditions (described for example, in Saiki, et al. (1988) Science 239:487-491), which result in specific amplification of sequences of PCVII or its variants.

The term "functionally equivalent" intends that the amino acid sequence of a protein is one that will elicit a substantially equivalent or enhanced immunological P:IOPERjfna.SpciTicaiionsl764379-200 120 DIV.doc- 25/0903 O response, as defined above, as compared to the response elicited by a reference Samino acid sequence, or an immunogenic portion thereof.

A "heterologous" region of a DNA construct is an identifiable segment of DNA within or attached to another DNA molecule that is not found in association with 0 the other molecule in nature. Thus, when the heterologous region encodes a viral 00 Sgene, the gene will usually be flanked by DNA that does not flank the viral gene in 0 the genome of the source virus. Another example of the heterologous coding sequence is a construct where the coding sequence itself is not found in nature synthetic sequences having codons different from the native gene). Allelic variation or naturally occurring mutational events do not give rise to a heterologous region of DNA, as used herein.

The term "treatment" as used herein refers to either the prevention of infection or reinfection (prophylaxis), or (ii) the reduction or elimination of symptoms of the disease of interest (therapy).

As used herein, a "biological sample" refers to a sample of tissue or fluid isolated from a subject, including but not limited to, for example, blood, plasma, serum, fecal matter, urine, bone marrow, bile, spinal fluid, lymph tissue and lymph fluid, samples of the skin, external secretions of the skin, respiratory, intestinal, and genitourinary tracts, tears, saliva, milk, blood cells, organs, biopsies and also samples of in vitro cell culture constituents including but not limited to conditioned media resulting from the growth of cells and tissues in culture medium, e.g., recombinant cells, and cell components.

As used herein, the terms "label" and "detectable label" refer to a molecule capable of detection, including, but not limited to, radioactive isotopes, fluorescers, chemiluminescers, enzymes, enzyme substrates, enzyme cofactors, enzyme inhibitors, chromophores, dyes, metal ions, metal sols, ligands biotin or haptens) and the like. The term "fluorescer" refers to a substance or a portion thereof which is capable of exhibiting fluorescence in the detectable range.

Particular examples of labels which may be used under the invention include P-.%OPERjrmSpciricainl)7900202120 DIV.doc. 25/9/03 O fluorescein, rhodamine, dansyl, umbelliferone, Texas red, luminol, NADPH and a- 3p galactosidase.

By "vertebrate subject" is meant any member of the subphylum cordata, including, without limitation, mammals such as cattle, sheep, pigs, goats, horses, and man; domestic animals such as dogs and cats; and birds, including domestic, wild and game birds such as cocks and hens including chickens, turkeys and other Sgallinaceous birds. The term does not denote a particular age. Thus, adult and Snewborn animals, as well as fetuses, are intended to be covered.

c[ B. General Methods Central to the present invention is the discovery of a new circovirus termed "PCVII" herein, isolated from PMWS-affected pigs. The useful materials and processes of the present invention are made possible by the discovery of a family of nucleotide sequences, each containing an entire genome of a novel PCVII virus.

The availability of this family of polynucleotides, first, permits the isolation of other members of the genome family which differ by small heterogeneities. Second, it permits the construction of DNA fragments and proteins useful in diagnosis. For example, oligomers of at least about 8-10 nucleotides or more, preferably, oligomers comprising at least about 15-20 nucleotides, are useful as hybridization probes in disease diagnosis. Such probes may be used to detect the presence of the viral genome in, for example, sera of subjects suspected of harboring the virus.

Similarly, the genes encoding the proteins can be cloned and used to design probes to detect and isolate homologous genes in other viral isolates.

The PCVII sequences also allow the design and production of PCVII-specific polypeptides which are useful as diagnostic reagents for the presence of antibodies raised against PCVII in serum or blood. Antibodies against these polypeptides are also useful as diagnostics. Because several open reading frames can be deciphered in the context of the complete genome, the primary structures of PCVII-related proteins can be deduced. Finally, knowledge of the gene sequences also enables the design and production of vaccines effective against P:%OPERkjmnSpceiica,.ons764379.2O22 I20 DIV.doc- 25/9/03 O PCVII and hence useful for the prevention of PMWS and also for the production of Sprotective antibodies.

Sequencing information available from the genome allows the amino acid s sequence of the various polypeptides encoded by the viral genome to be deduced and suitable epitopes identified. The full-length proteins encoded by the several 00oo ORFs identified in the PCVII genome, or suitable portions thereof, can be Sproduced using fragments of the relevant DNA which are obtained and expressed °independently, thus providing desired polypeptides using recombinant techniques.

to Both procaryotic and eucaryotic hosts are useful for such expression. Short polypeptide fragments may also be chemically synthesized and linked to carrier proteins for use as vaccines. In addition, epitopes may be produced linked to a protein conferring immunogenicity. The proteins thus produced may themselves be used as vaccines, or may be used to induce immunocompetent B cells in hosts, which B cells can then be used to produce hybridomas that secrete antibodies useful in passive immunotherapy.

The complete genetic sequences for three isolates of PCVII, PCVII 412 (SEQ ID NO:7), PCVII 9741 (SEQ ID NO:17), AND PCVII B9 (SEQ ID NO:18 30), are shown in FIGS. 11A-11B. The percent nucleotide sequence homologies among the various isolates of PCVII are more than 99% identical. The newly discovered viral genome shares approximately 76% identity with PCV isolated from infected cells at the nucleotide level (termed "PCVI" herein). As described further in the examples, nucleotide insertions and deletions (indels) have been found in three regions.

As shown in FIG. 8, the new virus contains at least six potential open reading frames (ORFs) encoding proteins comprising more than 50 amino acid residues, while PCVI derived from PK15 has seven potential ORFs. The ORFs for representative PCVII isolates occur at the following nucleotide positions, using the numbering of the PCVII isolates shown in FIGS. 11A-11B: ORF 1 51 to 992 ORF 2 1735 to 1037 P.AOPERjrrpecifcaions%764379200232 120 DIV.doc 21/9103

NO

0 ORF 3 671 to 360 ORF 4 565 to 389 ORF 5 553 to 729 ORF 6 1016 to 1174 (the nucleotide sequences 1-6 as defined above do not include the stop codon).

00 SThe polypeptides encoded by the six ORFs are shown in FIGS. 9A-9C.

0 The main cellular targets for PCVII are mononuclear cells in the peripheral blood, 0 to likely macrophage cells, although the virus is also found in various tissues and organs in infected animals. The affected macrophages lose their normal function, causing damage to the host immune system, leading to death.

The cloning and sequencing of the novel circoviruses has provided information about the causative agent of PMWS. As explained above, the sequencing information, as well as the clones and its gene products, are useful for diagnosis and in vaccine development. In particular, PCR and antibody-based diagnostic methods are useful in the diagnosis of the disease and were used herein to specifically identify and differentiate this novel PCVII virus from PCVI derived from persistently infected PK15 cells. The sequencing information is also useful in the design of specific primers, to express viral-specific gene products, to study the viral structure, to generate specific antibodies and to identify virulent genes in porcine circovlrus-related diseases.

B.1. Preparation of the PCVII Gene Sequence The new viral genome of PCVII 412 were obtained from viruses isolated from tissue of PMWS-affected pigs. Viral DNA was extracted from variable sources, including pellets of infected Dulac and Vero cells, peripheral blood buffy-coat cells, tissues from infected animals and serum. DNA was extracted from the samples using techniques discussed more fully in the examples.

By comparing the sequence and structural similarity among the known viruses in P.%OPERjrmsSpecirlci conb74S79.200O3O2 120 OIV.doc 2Y9/03 the circovirus family, a unique primer was designed taking advantage of the complementary sequences of a conserved stem loop structure. One-primer PCR Swas then performed and the products cloned. Two full-length viral genomes in Sdifferent orientations inserted into a plasmid vector were completely sequenced in both directions. Additional PCR products were made and sequenced to ensure the fidelity of the primer/stem loop region.

00 SUsing similar primers, other PCVII isolates, including PCVII 9741, and PCVII B9, I0 were obtained. This appears to be the first time a circovirus has been cloned ilo from viral particles instead of from a replicated form of DNA.

The description of the method to retrieve the PCVII genome is, of course, mostly of historical interest. The resultant sequence is provided herein, and the entire sequence, or any portion thereof, could also be prepared using synthetic methods, or by a combination of synthetic methods with retrieval of partial sequences using methods similar to those here described.

B.2. Production of PCVII Proteins The availability of PCVII genomic sequences permits construction of expression vectors encoding viral polypeptides and antigenically active regions thereof, derived from the PCVII genome. Fragments encoding the desired proteins can be obtained from cDNA clones using conventional restriction digestion or by synthetic methods and are ligated into vectors, for example, containing portions of fusion sequences such as .beta.-galactosidase. Any desired portion of the PCVII genome containing an open reading frame can be obtained as a recombinant protein, such as a mature or fusion protein, or can be provided by-chemical synthesis or general recombinant means.

It is readily apparent that PCVII proteins encoded by the above-described DNA sequences, active fragments, analogs and chimeric proteins derived from the same, can be produced by a variety of methods. Recombinant products can take the form of partial protein sequences, full-length sequences, precursor forms that P:\OPERjnsSpecirlcaiionA7643792002302120 DIVAdc -2S103 include signal sequences, mature forms without signals, or even fusion proteins with an appropriate leader for the recombinant host, or with another subunit Santigen sequence for another pathogen).

Gene libraries can be constructed and the resulting clones used to transform an 0 appropriate host cell. Colonies can be pooled and screened using polyclonal serum or monoclonal antibodies to the PCVII protein.

0Alternatively, once the amino acid sequences are determined, oligonucleotide probes which contain the codons for a portion of the determined amino acid sequences can be prepared and used to screen genomic or cDNA libraries for genes encoding the subject proteins. The basic strategies for preparing oligonucleotide probes and DNA libraries, as well as their screening by nucleic acid hybridization, are well known to those of ordinary skill in the art. See, e.g., DNA Cloning: Vol. I, supra; Nucleic Acid Hybridization, supra; Oligonucleotide Synthesis, supra; Sambrook et al., supra. Once a clone from the screened library has been identified by positive hybridization, it can be confirmed by restriction enzyme analysis and DNA sequencing that the particular library insert contains a PCVII protein gene or a homolog thereof. The genes can then be further isolated using standard techniques and, if desired, PCR approaches or restriction enzymes employed to delete portions of the full-length sequence.

Similarly, genes can be isolated directly from viruses using known techniques, such as phenol extraction and the sequence further manipulated to produce any desired alterations. See, the examples herein and Hamel et al. (1998) J.

Virol. 72:5262-5267, for a description of techniques used to obtain and isolate viral

DNA.

Alternatively, DNA sequences can be prepared synthetically rather than cloned.

The DNA sequences can be designed with the appropriate codons for the particular amino acid sequence if the sequences are to be used in protein production. In general, one will select preferred codons for the intended host if the sequence will be used for expression. The complete sequence is assembled from P:'OPF.R\jnmskSpcirlcnaions764370.202302 20 DIVdo 25/9103 Soverlapping oligonucleotides prepared by standard methods and assembled into a complete coding sequence. See, Edge (1981) Nature 292:756; Nambair et al.

S(1984) Science 223:1299; Jay et al. (1984) J. Biol. Chem. 259:6311.

Once coding sequences for the desired proteins have been prepared or isolated, 0 they can be cloned into any suitable vector or replicon. Numerous cloning vectors 00 are known to those of skill in the art, and the selection of an appropriate cloning 0vector is a matter of choice. Examples of recombinant DNA vectors for cloning and 0host cells which they can transform include the bacteriophage X coli), pBR322 S o10 coli), pACYC177 coli), pKT230 (gram-negative bacteria), pGV1106 (gramnegative bacteria), pLAFR1 (gram-negative bacteria), pME290 (non-E. coli gramnegative bacteria), pHV14 coli and Bacillus subtilis), pBD9 (Bacillus), plJ61 (Streptomyces), pUC6 (Streptomyces), YIp5 (Saccharomyces), YCp19 (Saccharomyces) and bovine papilloma virus (mammalian cells). See, Sambrook et al., supra; DNA Cloning, supra; B. Perbal, supra.

The gene can be placed under the control of a promoter, ribosome binding site (for bacterial expression) and, optionally, an operator (collectively referred to herein as "control" elements), so that the DNA sequence encoding the desired protein is transcribed into RNA in the host cell transformed by a vector containing this expression construction. The coding sequence may or may not contain a signal peptide or leader sequence. If a signal sequence is included, it can either be the native, homologous sequence, or a heterologous sequence. Leader sequences can be removed by the host in post-translational processing. See, U.S. Pat.

Nos. 4,431,739; 4,425,437; 4,338,397.

Other regulatory sequences may also be desirable which allow for regulation of expression of the protein sequences relative to the growth of the host cell.

Regulatory sequences are known to those of skill in the art, and examples include those which cause the expression of a gene to be turned on or off in response to a chemical or physical stimulus, including the presence of a regulatory compound.

Other types of regulatory elements may also be present in the vector, for example, enhancer sequences.

P:%OPFRljrm\SpcciiciionsA7643792002.O2 120 DIV.dc 2519/03 SThe control sequences and other regulatory sequences may be ligated to the coding sequence prior to insertion into a vector, such as the cloning vectors described above. Alternatively, the coding sequence can be cloned directly into an expression vector which already contains the control sequences and an 0 appropriate restriction site.

oo In some cases it may be necessary to modify the coding sequence so that it may be attached to the control sequences with the appropriate orientation; to maintain the proper reading frame. It may also be desirable to produce mutants or analogs of the desired PCVII protein. Mutants or analogs may be prepared by the deletion of a portion of the sequence encoding the protein, by insertion of a sequence, and/or by substitution of one or more nucleotides within the sequence.

Techniques for modifying nucleotide sequences, such as site-directed mutagenesis, are described in, Sambrook et al., supra; DNA Cloning, supra; Nucleic Acid Hybridization, supra.

The expression vector is then used to transform an appropriate host cell. A number of mammalian cell lines are known in the art and include immortalized cell lines available from the American Type Culture Collection (ATCC), such as, but not limited to, Chinese hamster ovary (CHO) cells, HeLa cells, baby hamster kidney (BHK) cells, monkey kidney cells (COS), human hepatocellular carcinoma cells Hep G2), Madin-Darby bovine kidney ("MDBK") cells, as well as others.

Similarly, bacterial hosts such as E. coli, Bacillus subtilis, and Streptococcus spp., will find use with the present expression constructs. Yeast hosts useful in the present invention include inter alia, Saccharomyces cerevisiae, Candida albicans, Candida maltosa, Hansenula polymorpha, Kluyveromyces fragilis, Kluyveromyces lactis, Pichia guillerimondii, Pichia pastoris, Schizosaccharomyces pombe and Yarrowia lipolytica. Insect cells for use with baculovirus expression vectors include, inter alia, Aedes aegypti, Autographa californica, Bombyx mori, Drosophila melanogaster, Spodoptera frugiperda, and Trichoplusia ni.

Depending on the expression system and host selected, the proteins of the P:IOPERl.mjSpcilcrnions%7643792002302 120 DIV.doc 259103 present invention are produced by culturing host cells transformed by an Sexpression vector described above under conditions whereby the protein of ;Z interest is expressed. The protein is then isolated from the host cells and purified.

If the expression system secretes the protein into the growth media, the protein can be purified directly from the media. If the protein is not secreted, it is isolated Sfrom cell lysates. The selection of the appropriate growth conditions and recovery methods are within the skill of the art.

0 The proteins of the present invention may also be produced by chemical synthesis 0 to such as solid phase peptide synthesis, using known amino acid sequences or amino acid sequences derived from the DNA sequence of the genes of interest.

Such methods are known to those skilled in the art. See, J. M. Stewart and J.

D. Young, Solid Phase Peptide Synthesis, 2nd Ed., Pierce Chemical Co., Rockford, III. (1984) and G. Barany and R. B. Merrifield, The Peptides: Analysis, Synthesis, Biology, editors E. Gross and J. Meienhofer, Vol. 2, Academic Press, New York, (1980), pp. 3-254, for solid phase peptide synthesis techniques; and M.

Bodansky, Principles of Peptide Synthesis, Springer-Verlag, Berlin (1984) and E.

Gross and J. Meienhofer, Eds., The Peptides: Analysis, Synthesis, Biology, supra, Vol. 1, for classical solution synthesis. Chemical synthesis of peptides may be preferable if a small fragment of the antigen in question is capable of raising an immunological response in the subject of interest.

Analysis of the genome shows the presence of at least six open reading frames, at least one of which encodes the putative DNA replicase gene.

B.3. Preparation of Antigenic Polypeptides and Conjugation with Carrier The antigenic region of peptides is generally relatively small--typically 10 amino acids or less in length. Fragments of as few as 5 amino acids may typically characterize an antigenic region. Accordingly, using the genome of PCVII as a basis, DNAs encoding short segments of polypeptides, derived from any of the various ORFs of PCVII, such as ORFs 1-6, and particularly ORF 2, can be expressed recombinantly either as fusion proteins or as isolated peptides. In P:OPCRjrriuSpcciicnlionsx76437920202 120 DIVdoc. 259/03 addition, short amino acid sequences can be chemically synthesized. In instances wherein the synthesized peptide is correctly configured so as to provide the Scorrect epitope, but too small to be immunogenic, the peptide may be linked to a O suitable carrier.

SA number of techniques for obtaining such linkage are known in the art, including 00 the formation of disulfide linkages using N-succinimidyl-3-(2-pyridyl-thio)propionate S(SPDP) and succinimidyl 4-(N-maleimido-methyl)cyclohexane-1-carboxylate 0 (SMCC) obtained from Pierce Company, Rockford, III. (If the peptide lacks a 0 10 sulfhydryl, this can be provided by addition of a cysteine residue.) These reagents create a disulfide linkage between themselves and peptide cysteine residues on one protein and an amide linkage through the E-amino on a lysine, or other free amino group in the other. A variety of such disulfide/amide-forming agents are known. See, for example, Immun. Rev. (1982) 62:185. Other bifunctional coupling agents form a thioether rather than a disulfide linkage. Many of these thioetherforming agents are commercially available-and include reactive esters of 6maleimidocaproic acid, 2-bromoacetic acid, 2-iodoacetic acid, 4-(N-maleimidomethyl)cyclohexane-1-carboxylic acid, and the like. The carboxyl groups can be activated by combining them with succinimide or 1-hydroxy-2-nitro-4-sulfonic acid, sodium salt. The foregoing list is not meant to exhaustive, and modifications of the named compounds can clearly be used.

Any carrier may be used, which does not itself induce the production of antibodies harmful to the host, such as the various serum albumins, tetanus toxoids, or keyhole limpet hemocyanin (KLH).

The conjugates, when injected into suitable subjects, will result in the production of antisera which contain immunoglobulins specifically reactive against not only the conjugates, but also against fusion proteins carrying the analogous portions of the sequence, and against appropriate determinants within whole PCVII.

B.4. Production of Antibodies P:\OPER\jr\Spccificalions\7M6379-2002302120 DIV.doc 25/9/03 SProteins encoded by the novel viruses of the present invention, or their fragments, can be used to produce antibodies, both polyclonal and monoclonal. If polyclonal antibodies are desired, a selected mammal, mouse, rabbit, goat, horse, etc.) is immunized with an antigen of the present invention, or its fragment, or a O mutated antigen. Serum from the immunized animal is collected and treated 00 according to known procedures. See, Jurgens et al. (1985) J. Chrom.

348:363-370. If serum containing polyclonal antibodies is used, the polyclonal antibodies can be purified by immunoaffinity chromatography, using known procedures.

Monoclonal antibodies to the proteins and to the fragments thereof, can also be readily produced by one skilled in the art. The general methodology for making monoclonal antibodies by using hybridoma technology is well known. Immortal antibody-producing cell lines can be created by cell fusion, and also by other techniques such as direct transformation of B lymphocytes with oncogenic DNA, or transfection with Epstein-Barr virus. See, M. Schreier et al., Hybridoma Techniques (1980); Hammerling et al., Monoclonal Antibodies and T-cell Hybridomas (1981); Kennett et al., Monoclonal Antibodies (1980); see also U.S.

Pat. Nos. 4,341,761; 4,399,121; 4,427,783; 4,444,887; 4,452,570; 4,466,917; 4,472,500, 4,491,632; and 4,493,890. Panels of monoclonal antibodies produced against the desired protein, or fragment thereof, can be screened for various properties; for isotype, epitope, affinity, etc. Monoclonal antibodies are useful in purification, using immunoaffinity techniques, of the individual antigens which they are directed against. Both polyclonal and monoclonal antibodies can also be used for passive immunization or can be combined with subunit vaccine preparations to enhance the immune response. Polyclonal and monoclonal antibodies are also useful for diagnostic purposes.

B.5. Vaccine Formulations and Administration The novel viral proteins of the present invention can be formulated into vaccine compositions, either alone or in combination with other antigens, for use in P:%OPER\jrm\Sprcticrnaons764379.2002302 120 DIV.c 25/9/03 immunizing subjects as described below. Methods of preparing such formulations are described in, Remington's Pharmaceutical Sciences, Mack Publishing SCompany, Easton, Pennsylvania, 18 Edition, 1990. Typically, the vaccines of the present invention are prepared as injectables, either as liquid solutions or suspensions. Solid forms suitable for solution in or suspension in liquid vehicles O prior to injection may also be prepared. The preparation may-also be emulsified or oo t the active ingredient encapsulated in liposome vehicles. The active immunogenic O ingredient is generally mixed with a compatible pharmaceutical vehicle, such as, N for example, water, saline, dextrose, glycerol, ethanol, or the like, and combinations thereof. In addition, if desired, the vehicle may contain minor amounts of auxiliary substances such as wetting or emulsifying agents and pH buffering agents.

Adjuvants which enhance the effectiveness of the vaccine may also be added to the formulation. Such adjuvants include, without limitation, adjuvants formed from aluminum salts (alum), such as aluminum hydroxide, aluminum phosphate, aluminum sulfate, etc; oil-in-water and water-in-oil emulsion formulations, such as Complete Freunds Adjuvant (CFA), Incomplete Freunds Adjuvant (IFA), avridine and dimethyldioctadecyl ammonium bromide (DDA); adjuvants formed from bacterial cell wall components such as adjuvants including monophosphoryl lipid A (MPL) (Imoto et al. (1985) Tet. Lett. 26:1545-1548), trehalose dimycolate (TDM), and cell wall skeleton (CWS); adjuvants derived from ADP-ribosylating bacterial toxins, such as derived from diphtheria toxin (for example, CRM.sub.197, a nontoxic diphtheria toxin mutant (see, Bixler et al. (1989) Adv. Exp. Med. Biol.

251:175; and Constantino et al. (1992) Vaccine), pertussis toxin cholera toxin the E. coli heat-labile toxins (LT1 and LT2), Pseudomonas endotoxin A, C. botulinum C2 and C3 toxins, as well as toxins from C. perfringens, C. spiriforma and C. difficile; saponin adjuvants such as Quil A Pat. No. 5,057,540), or particles generated from saponins such as ISCOMs (immunostimulating complexes); cytokines, such as interleukins IL-1, IL-2, IL-4, IL-5, IL-6, IL-7, IL-12, etc.), interferons gamma interferon), macrophage colony stimulating factor (M-CSF), tumor necrosis factor (TNF), etc; muramyl peptides such as Nacetyl-muramyl-L-threonyl-D-isoglutamine (thr-MDP), N-acetyl-normuramyl

L

P:%OPER~jrmsSpecirlcionsl7643792OO2 I20 DIVdoc- 25/9103 Salanyl- -isoglutamine (nor-MDP), N-acetylmuramyl-L-alanyl-.sup.Disoglutaminyl-L alanine-2-(l'-2'-dipalmitoyl-sn-glycero-3 h Shydroxyphosphoryloxy)-ethylamine (MTP-PE), etc.; adjuvants derived from the CpG family of molecules, CpG dinucleotides and synthetic oligonucleotides which gomprise CpG motifs (see, Krieg et al. Nature (1995) 374:546 and Davis et 0al. J. Immunol. (1998) 160:870-876); and synthetic adjuvants such as PCPP (Poly 00 di(carboxylatophenoxy)phosphazene) (Payne et al. Vaccines (1998) 16:92-98).

O Such adjuvants are commercially available from a number of distributors such as °Accurate Chemicals; Ribi Immunechemicals, Hamilton, MT; GIBCO; Sigma, St.

S 10 Louis, Mo.

As explained above, the proteins may be linked to a carrier in order to increase the immunogenicity thereof. Suitable carriers include large, slowly metabolized macromolecules such as proteins, including serum albumins, keyhole limpet hemocyanin, immunoglobulin molecules, thyroglobulin, ovalbumin, and other proteins well known to those skilled in the art; polysaccharides, such as sepharose, agarose, cellulose, cellulose beads and the like; polymeric amino acids such as polyglutamic acid, polylysine, and the like; amino acid copolymers; and inactive virus particles.

The proteins may be used in their native form or their functional group content may be modified by, for example, succinylation of lysine residues or reaction with Cysthiolactone. A sulfhydryl group may also be incorporated into the carrier (or antigen) by, for example, reaction of amino functions with 2-iminothiolane or the Nhydroxysuccinimide ester of 3-(4-dithiopyridyl propionate. Suitable carriers may also be modified to incorporate spacer arms (such as hexamethylene diamine or other bifunctional molecules of similar size) for attachment of peptides.

Other suitable carriers for the proteins of the present invention include VP6 polypeptides of rotaviruses, or functional fragments thereof, as disclosed in U.S.

Pat. No. 5,071,651, incorporated herein by reference. Also useful is a fusion product of a viral protein and the subject immunogens made by methods disclosed in U.S. Pat. No. 4,722,840. Still other suitable carriers include cells, such as P:%OPER~jryuSpecirications 764379 20 0210 DIV.doc- 25I/03 Slymphocytes, since presentation in this form mimics the natural mode of presentation in the subject, which gives rise to the immunized state. Alternatively, Sthe proteins of the present invention may be coupled to erythrocytes, preferably the subject's own erythrocytes. Methods of coupling peptides to proteins or cells are known to those of skill in the art.

00 oO Furthermore, the proteins may be formulated into vaccine compositions in either O neutral or salt forms. Pharmaceutically acceptable salts include the acid addition N salts (formed with the free amino groups of the active polypeptides) and which are S to formed with in-organic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric, mandelic, and the like. Salts formed from free carboxyl groups may also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, 2-ethylamino ethanol, histidine, procaine, and the like.

Vaccine formulations will contain a "therapeutically effective amount" of the active ingredient, that is, an amount capable of eliciting an immune response in a subject to which the composition is administered. Such a response will be demonstrated by either a reduction or lack of symptoms normally displayed by an infected host and/or a quicker recovery time.

The exact amount is readily determined by one skilled in the art using standard tests. The protein concentration will typically range from about 1% to about of the composition, or even higher or lower if appropriate.

To immunize a subject, the vaccine is generally administered parenterally, usually by intramuscular injection. Other modes of administration, however, such as subcutaneous, intraperitoneal and intravenous injection, are also acceptable. The quantity to be administered depends on the animal to be treated, the capacity of the animal's immune system to synthesize antibodies, and the degree of protection desired. Effective dosages can be readily established by one of ordinary skill in the art through routine trials establishing dose response curves. The subject P:%OPERkjs\Specicaont764379200 202120 DIV.doc. 25100) is immunized by administration of the vaccine in at least one dose, and preferably N two doses. Moreover, the animal may be administered as many doses as is required to maintain a state of immunity to infection.

Additional vaccine formulations which are suitable for other modes of 0 administration include suppositories and, in some cases, aerosol, intranasal, oral oo formulations, and sustained release formulations. For suppositories, the vehicle O composition will include traditional binders and carriers, such as, polyalkaline I glycols, or triglycerides. Such suppositories may be formed from mixtures o1 containing the active ingredient in the range of about 0.5% to about 10% preferably about 1% to about Oral vehicles include such normally employed excipients as, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium, stearate, sodium saccharin cellulose, magnesium carbonate, and the like. These oral vaccine compositions may be taken in the form of solutions, suspensions, tablets, pills, capsules, sustained release formulations, or powders, and contain from about 10% to about 95% of the active ingredient, preferably about 25% to about Intranasal formulations will usually include vehicles that neither cause irritation to the nasal mucosa nor significantly disturb ciliary function. Diluents such as water, aqueous saline or other known substances can be employed with the subject invention. The nasal formulations may also contain preservatives such as, but not limited to, chlorobutanol and benzalkonium chloride. A surfactant may be present to enhance absorption of the subject proteins by the nasal mucosa.

Controlled or sustained release formulations are made by incorporating the protein into carriers or vehicles such as liposomes, nonresorbable impermeable polymers such as ethylenevinyl acetate copolymers and Hytrel" copolymers, swellable polymers such as hydrogels, or resorbable polymers such as collagen and certain polyacids or polyesters such as those used to make resorbable sutures. The proteins can also be delivered using implanted mini-pumps, well known in the art.

P:NOPER~jfnacSpecirictionsN764379-20023120 OIV.dOc 25/9/03 The proteins of the instant invention can also be administered via a carrier virus which expresses the same. Carrier viruses which will find use with the instant Sinvention include but are not limited to the vaccinia and other pox viruses, adenovirus, and herpes virus. By way of example, vaccinia virus recombinants expressing the novel proteins can be constructed as follows. The DNA encoding 0 the particular protein is first inserted into an appropriate vector so that it is adjacent oo 7- to a vaccinia promoter and flanking vaccinia DNA sequences, such as the Ssequence encoding thymidine kinase This vector is then used to transfect N0 cells which are simultaneously infected with vaccinia. Homologous recombination 0 10 serves to insert the vaccinia promoter plus the gene encoding the instant protein into the viral genome. The resulting TK- recombinant can be selected by culturing the cells in the presence of 5-bromodeoxyuridine and picking viral plaques resistant thereto.

An alternative route of administration involves gene therapy or nucleic acid immunization. Thus, nucleotide sequences (and accompanying regulatory elements) encoding the subject proteins can be administered directly to a subject for in vivo translation thereof. Alternatively, gene transfer can be accomplished by transfecting the subject's cells or tissues ex vivo and reintroducing the transformed material into the host. DNA can be directly introduced into the host organism, i.e., by injection (see U.S. Pat. Nos. 5,580,859 and 5,589,466; International Publication No. WO/90/11092; and Wolff et al. (1990) Science 247:1465-1468). Liposomemediated gene transfer can also be accomplished using known methods. See, U.S. Pat. No. 5,703,055; Hazinski et al. (1991) Am. J. Respir. Cell Mol. Biol.

4:206-209; Brigham et al. (1989) Am. J. Med. Sci. 298:278-281; Canonico et al.

(1991) Clin. Res. 39:219A; and Nabel et al. (1990) Science 249:1285-1288.

Targeting agents, such as antibodies directed against surface antigens expressed on specific cell types, can be covalently conjugated to the liposomal surface so that the nucleic acid can be delivered to specific tissues and cells susceptible to infection.

B.6. Diagnostic Assays P: OPER~jrns\Speciriations%764379-200230120 DIV.c 2519/03 As explained above, the proteins of the present invention may also be used as diagnostics to detect the presence of reactive antibodies of PCVII in a biological O' sample in order to determine the presence of PCVII infection. For example, the presence of antibodies reactive with the proteins can be detected using standard 0 electrophoretic and immunodiagnostic techniques, including immunoassays such 00oo 0- as competition, direct reaction, or sandwich type assays. Such assays include, but 0are not limited to, Western blots; agglutination tests; enzyme-labeled and \0 mediated immunoassays, such as ELISAs; biotin/avidin type assays; 0 0 radioimmunoassays; immunoelectrophoresis; immunoprecipitation, etc. The reactions generally include revealing labels such as fluorescent, chemiluminescent, radioactive, enzymatic labels or dye molecules, or other methods for detecting the formation of a complex between the antigen and the antibody or antibodies reacted therewith.

The aforementioned assays generally involve separation of unbound antibody in a liquid phase from a solid phase support to which antigen-antibody complexes are bound. Solid supports which can be used in the practice of the invention include substrates such as nitrocellulose in membrane or microtiter well form); polyvinylchloride sheets or microtiter wells); polystyrene latex beads or microtiter plates); polyvinylidine fluoride; diazotized paper; nylon membranes; activated beads, magnetically responsive beads, and the like.

Typically, a solid support is first reacted with a solid phase component one or more PCVII proteins) under suitable binding conditions such that the component is sufficiently immobilized to the support. Sometimes, immobilization of the antigen to the support can be enhanced by first coupling-the antigen to a protein with better binding properties. Suitable coupling proteins include, but are not limited to, macromolecules such as serum albumins including bovine serum albumin (BSA), keyhole limpet hemocyanin, immunoglobulin molecules, thyroglobulin, ovalbumin, and other proteins well known to those skilled in the art. Other molecules that can be used to bind the antigens to the support include polysaccharides, polylactic acids, polyglycolic acids, polymeric amino acids, amino acid copolymers, and the P:%OPERjna\Speirications%76 379-200230120 01%.dc 299103 like. Such molecules and methods of coupling these molecules to the antigens, are well known to those of ordinary skill in the art. See, Brinkley, M. A.

SBioconjugate Chem. (1992) 3:2-13; Hashida et al., J. Appl. Biochem. (1984) 6:56- 63; and Anjaneyulu and Staros, International J. of Peptide and Protein Res. (1987) 30:117-124.

0 0 After reacting the solid support with the solid phase component, any non- 0immobilized solid-phase components are removed from the support by washing, \0 and the support-bound component is then contacted with a biological sample io suspected of containing ligand moieties antibodies toward the immobilized antigens) under suitable binding conditions. After washing to remove any nonbound ligand, a secondary binder moiety is added under suitable binding conditions, wherein the secondary binder is capable of associating selectively with the bound ligand. The presence of the secondary binder can then be detected using techniques well known in the art.

More particularly, an ELISA method can be used, wherein the wells of a microtiter plate are coated with a desired protein. A biological sample containing or suspected of containing anti-protein immunoglobulin molecules is then added to the coated wells. After a period of incubation sufficient to allow antibody binding to the immobilized antigen, the plate(s) can be washed to remove unbound moieties and a detectably labeled secondary binding molecule added. The secondary binding molecule is allowed to react with any captured sample antibodies, the plate washed and the presence of the secondary binding molecule detected using methods well known in the art.

Thus, in one particular embodiment, the presence of bound anti-antigen ligands from a biological sample can be readily detected using a secondary binder comprising an antibody directed against the antibody ligands. A number of antiporcine immunoglobulin (Ig) molecules are known in the art which can be readily conjugated to a detectable enzyme label, such as horseradish peroxidase, alkaline phosphatase or urease, using methods known to those of skill in the art. An appropriate enzyme substrate is then used to generate a detectable signal. In P:IOPERljrmSpeciricoions764379.2OO2O2 I20 DIV.dc.- 25Aq/03 other related embodiments, competitive-type ELISA techniques can be practiced Susing methods known to those skilled in the art.

Assays can also be conducted in solution, such that the proteins and antibodies specific for those proteins form complexes under precipitating conditions. In one Sparticular embodiment, proteins can be attached to a solid phase particle an agarose bead or the like) using coupling techniques known in the art, such as by Sdirect chemical or indirect coupling. The antigen-coated particle is then contacted Sunder suitable binding conditions with a biological sample suspected of containing S 10 antibodies for the proteins. Cross-linking between bound antibodies causes the formation of particle-antigen-antibody complex aggregates which can be precipitated and separated from the sample using washing and/or centrifugation.

The reaction mixture can be analyzed to determine the presence or absence of antibody-antigen complexes using any of a number of standard methods, such as those immunodiagnostic methods described above.

In yet a further embodiment, an immunoaffinity matrix can be provided, wherein a polyclonal population of antibodies from a biological sample suspected of containing antibodies to the protein of interest is immobilized to a substrate. In this regard, an initial affinity purification of the sample can be carried out using immobilized antigens. The resultant sample preparation will thus only contain anti- PCVII moieties, avoiding potential nonspecific binding properties in the affinity support. A number of methods of immobilizing immunoglobulins (either intact or in specific fragments) at high yield and good retention of antigen binding activity are known in the art. Not being limited by any particular method, immobilized protein A or protein G can be used to immobilize immunoglobulins.

Accordingly, once the immunoglobulin molecules have been immobilized to provide an immunoaffinity matrix, labeled proteins are contacted with the bound antibodies under suitable binding conditions. After any non-specifically bound antigen has been washed from the immunoaffinity support, the presence of bound antigen can be determined by assaying for label using methods known in the art.

P:OPERljnplecflcric.ons1643792002302 120 DIVdoc. 2519103 Additionally, antibodies raised to the proteins, rather than the proteins themselves, can be used in the above-described assays in order to detect the presence of antibodies to the proteins in a given sample. These assays are performed essentially as described above and are well known to those of skill in the art.

00

CO

Furthermore, nucleic acid-based assays may also be conducted. In this regard, Susing the disclosed PCVII nucleic acid sequences as a basis, oligomers can be Sprepared which are useful as hybridization probes or PCR primers to detect the to presence of the viral genome in, for example, biological samples from subjects suspected of harboring the virus. Oligomers for use in this embodiment of the invention are approximately 8 nucleotides or more in length, preferably at least about 10-12 nucleotides in length, more preferably at least about 15 to nucleotides in length and up to 50 or more nucleotides in length. Preferably, the oligomers derive from regions of the viral genome which lack heterogeneity.

The oligomers are prepared either by excision from the genome, or recombinantly or synthetically. For example, the oligomers can be prepared using routine methods, such automated oligonucleotide synthetic methods.

The oligomers may be used as probes in diagnostic assays. In a representative assay, the biological sample to be analyzed is treated to extract the nucleic acids contained therein. The resulting nucleic acid from the sample may be subjected to gel electrophoresis or other size separation techniques. Alternatively, the nucleic acid sample may be dot-blotted without size separation. The probes are then labeled with a reporter moiety. Suitable labels, and methods for labeling probes, are known in the art and include, for example, radioactive labels incorporated by nick translation or kinasing, biotin, fluorescent probes and chemiluminescent probes. The nucleic acids extracted from the sample are then treated with the labeled probe under hybridization conditions of suitable stringencies.

The probes can be made completely complementary to the targeted PCVII gene sequence. However, when longer probes are used in the diagnostic assays, the P:'OPER'jrrm.Spcirclcions76437920023OI 20 DIV.oc 29/03 amount of complementarity may be less. Generally, conditions of high stringency are used in the assay methods, especially if the probes are completely or highly Scomplementary. However, lower stringency conditions should be used when targeting regions of heterogeneity. Methods of adjusting stringency are well known in the art. Such adjustments are made during hybridization and the washing 0 procedure and include adjustments to temperature, ionic strength, concentration of 00 formamide and length of time of the reaction. These factors are outlined in, e.g., 0 Sambrook et al., supra.

O

So0 In a more specific embodiment, the above-described method includes the use of PCVII nucleic acid specific probes where two probes (primers) define an internal region of the PCVII genome. In this embodiment, each probe has one strand containing a 3'-end internal to the PCVII nucleic acid internal region. The nucleic acid/probe hybridization complexes are then converted to double-strand probe containing fragments by primer extension reactions. Probe-containing fragments are amplified by successively repeating the steps of denaturing the doublestranded fragments to produce single-stranded fragments, (ii) hybridizing the single strands with the probes to form strand/probe complexes, (iii) generating double-stranded fragments from the strand/probe complexes in the presence of DNA polymerase and all four deoxyribonucleotides, and (iv) repeating steps to (iii) until a desired degree of amplification has been achieved. Amplification products are then identified according to established procedures. The method of the invention may further include a third polynucleotide probe capable of selectively hybridizing to the internal region described above but not to the specific probe/primer sequences used for amplification.

PCR techniques, such as those described above, are well known in the art. See, PCR Protocols: A Guide to Methods and Applications (Academic Press); PCR A Practical Approach (IRL press); Saiki et al. (1986) Nature 324:163.

Other amplification methods can also be used in the nucleic acid-based assays, such as ligase chain reaction (LCR), PCR, Q-beta replicase, and the like.

P:IOPERjmsSpcitr~ions764379-2002302 120 DIVckoc -25/9'03 Other assays for use herein include the "Bio-Bridge" system which uses terminal Sdeoxynucleotide transferase to add unmodified 3'-poly-dT-tails to a nucleic acid probe (Enzo Biochem. Corp.). The poly dt-tailed probe is hybridized to the target nucleotide sequence, and then to a biotin-modified poly-A. Additionally, EP 0124221 describes a DNA hybridization assay wherein the analyte is annealed to a 00 single-stranded DNA probe that is complementary to an enzyme-labelled O oligonucleotide, and the resulting tailed duplex is hybridized to an enzyme-labelled N oligonucleotide. EP 204510 describes a DNA hybridization assay in which analyte 0 0o DNA is contacted with a probe that has a tail, such as a poly-dT-tail, an amplifier strand that has a sequence that hybridizes to the tail of the probe, such as a poly- A sequence, and which is capable of binding a plurality of labelled strands. The technique first may involve amplification of the target PCVII sequences in sera to approximately 106 sequences/ml, as described above. The amplified sequence(s) then may be detected using a hybridization assay known in the art.

Furthermore, nucleic acid sequences derived from the PCVII viral genome, may also be used for in situ hybridization assays. Generally, such assays employ formalin-fixed cell culture preparations or tissues, such as lymph node, spleen, tonsil, liver, lung, heart, kidney, pancreas, nasal turbinate, large and small intestine, and the like. See, Sirinarumitr et al. (1996) J. Virol. Meth. 56:149- 160, for a description of a suitable in situ hybridization assay.

The above-described assay reagents, including the proteins, antibodies thereto or oligomers, can be provided in kits, with suitable instructions and other necessary reagents, in order to conduct immunoassays as described above. The kit can also contain, depending on the particular immunoassay used, suitable labels and other packaged reagents and materials wash buffers and the like). Standard immunoassays, such as those described above, can be conducted using these kits.

Below are examples of specific embodiments for carrying out the present P:'OPERjms'Specir~arnons764379O2302 120 DIV.c. 25/9/03 invention. The examples are offered for illustrative purposes only, and are not intended to limit the scope of the present invention in any way.

0BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1: DNA sequence of the genome of the Imp. 1011-48121 strain SFIG. 2: DNA sequence of the genome of the Imp. 1011-48285 strain oo 0 FIG. 3: DNA sequence of the genome of the Imp. 999 strain SFIG. 4: DNA sequence of the genome of the Imp. 1010 strain \0 FIG. 5: Alignment of the 4 sequences according to FIGS. 1 to 4 with the sequence of the PCV PK/15 strain FIG. 6: DNA sequence of the genome of the Imp. 999 strain as defined in the first filing in France on Oct. 3, 1997 FIG. 7: Alignments of the sequence of FIG. 6 with the sequence of the strain FIG. 8 is a diagram of PCVII 412, showing the location of open reading frames.

FIGS. 9A-9C depict the nucleotide sequence for the PCVII 412 genome (SEQ ID NO:7). Both senses are shown. The amino acid sequences corresponding to the translation products of the various ORFs are also shown as indicated: ORF 1 (SEQ ID NO:9); ORF 2 (SEQ ID NO:11); ORF 3 (SEQ ID ORF 4 (SEQ ID NO:13); ORF 5 (SEQ ID NO:26); and ORF 6 (SEQ ID NO:27).

FIGS. 10A-10D are comparisons of amino acid sequences from open reading frames of PCVII 412 versus corresponding open reading frames of PCVI isolated from PK15 cells.

FIG. 10A shows the amino acid sequence of ORF 1 of PCVII 412 (top line, SEQ ID NO:9) compared to the corresponding ORF from PCVI (bottom line, SEQ ID FIG. 10B shows the amino acid sequence of ORF 2 of PCVII 412 (top line, SEQ ID NO:11) compared to the corresponding ORF from PCVI (bottom line, SEQ ID NO:12).

FIG. 10C shows the amino acid sequence of ORF 4 of PCVII 412 (top line, SEQ ID NO:13) compared to the corresponding ORF from PCVI (bottom line, SEQ ID NO:14).

P: OPER'jsm%4S;pcciranons76437920232 20 DIV.doc 29/03 FIG. 10D shows the amino acid sequence of ORF 3 of PCVII 412 (top line, SEQ ID NO:15) compared to the corresponding ORF from PCVI (bottom line, SEQ ID NO:16).

FIGS. 11A-11B are comparisons of the nucleotide sequences of various PCV isolates: PCVI from PK15 cells (SEQ ID NO:8), PCVII 412 (SEQ ID NO:7), O PCVII 9741 (SEQ ID NO:17) and PCVII B9 (SEQ ID NO:18 00 FIG. 12 shows the results of multiplex PCR used for the detection of PCV O infection. The assay both identified PCV infection and distinguished ^0 between the presence of PCVI and PCVII. Lane 1 is a molecular weight S 10 marker. Lanes 2-4 are controls in the order of PCVII, PCVI and negative.

Lanes 5-13 are blood samples collected from piglets from a PMWS-affected herd.

FIG. 13 shows the results of multiplex PCR conducted on various tissue samples from a PMWS-affected piglet. Lane 1 in both rows is a molecular weight marker. Lane 2 in the top row is a positive PCVII control while lane 3 is a negative control. The remaining lanes are various tissue samples collected from the PMWS-affected piglet.

A EXPERIMENTAL FOR ISOLATES 999 1010 1011-48121 and 1011-48285 Example A 1 Culture and isolation of the porcine circovirus strains Tissue samples were collected in France, Canada and the USA from lung and lymph nodes of piglets. These piglets exhibited clinical signs typical of the postweaning multisystemic wasting syndrome. To facilitate the isolation of the viruses, the tissue samples were frozen at -70*C. immediately after autopsy.

For the viral isolation, suspensions containing about 15% tissue sample were prepared in a minimum medium containing Earle's salts (EMEM, BioWhittaker UK Ltd., Wokingham, UK), penicillin (100 IU/ml) and streptomycin (100 pg/ml) (MEM- SA medium), by grinding tissues with sterile sand using a sterile mortar and pestle.

This ground preparation was then taken up in MEM-SA, and then centrifuged at 3000 g for 30 minutes at in order to harvest the supernatant.

P:AOPER~jmSpecirc.ions%76379.2002302 120 DIV.doc 25/9/03 SPrior to the inoculation of the cell cultures, a volume of 100 pl of chloroform was added to 2 ml of each supernatant and mixed continuously for 10 minutes at room temperature. This mixture was then transferred to a microcentrifuge tube, centrifuged at 3000 g for 10 minutes, and then the supernatant was harvested.

O This supernatant was then used as inoculum for the viral isolation experiments.

00 O All the viral isolation studies were carried out on PK/15 cell cultures, known to be IN uncontaminated with the porcine circovirus (PCV), pestiviruses, porcine to adenoviruses and porcine parvoviruses (Allan G. et al Pathogenesis of porcine circovirus experimental infections of colostrum-deprived piglets and examination of pig foetal material. Vet. Microbiol. 1995, 44, 49-64).

The isolation of the porcine circoviruses was carried out according to the following technique: Monolayers of PK/15 cells were dissociated by trypsinization (with a trypsinversene mixture) from confluent cultures, and taken up in MEM-SA medium containing 15% foetal calf serum not contaminated by pestivirus (=MEM-G medium) in a final concentration of about 400,000 cells per ml. 10 ml aliquot fractions of this cell suspension were then mixed with 2 ml aliquot fractions of the inocula described above, and the final mixtures were aliquoted in 6 ml volumes in two Falcon flasks of 25 cm 2 These cultures were then incubated at +37 0 C for 18 hours under an atmosphere containing 10% CO 2 After incubation, the culture medium of the semi-confluent monolayers were treated with 300 mM D-glucosamine (Cat G48175, Sigma-Aldrich Company Limited, Poole, UK) (Tischr I. et al., Arch. Virol., 1987 96 39-57), then incubation was continued for an additional period of 48-72 hours at +37"C Following this last incubation, one of the two Falcons of each inoculum was subjected to 3 successive freeze/thaw cycles. The PK/15 cells of the remaining Falcon were treated with a trypsin-versene solution, resuspended in 20 ml of MEM-G medium, and then inoculated into 75 cm 2 Falcons at a concentration of 400,000 cells/ml.

P: OPrR~jjm\Speciricaiions%764379-200230120 DIVdoc.- 25/9/03 0 The freshly inoculated flasks were then "superinfected" by addition of 5 ml of the Scorresponding lysate obtained after the freeze/thaw cycles.

Example A 2 Preparation of the samples of cell culture for the detection of porcine circoviruses by immunofluorescence or by in situ hybridization 00 A volume of 5 ml of the "superinfected" suspension was collected and inoculated Sinto a Petri dish 55 mm in diameter containing a sterile and fat-free glass coverslip.

IN The cultures in the flasks and on glass coverslips were incubated at +37 0 C and S 10 treated with glucosamine as described in Example 1. The cultures on glass coverslips were 'harvested from 24 to 48 hours after the treatment with glucosamine and fixed, either with acetone for 10 minutes at room temperature, or with 10% buffered formaldehyde for 4 hours. Following this fixing, all the glass coverslips were stored at -70 0 C, on silica gel, before their use for the in situ hybridization studies and the immunocytochemical labelling studies.

Example A 3 Techniques for the detection of PCV sequences by in situ hybridization In situ hybridization was carried out on tissues collected from diseased pigs and fixed with formaldehyde and also on the preparations of cell cultures inoculated for the viral isolation (see Example 2) and fixed on glass coverslips.

Complete genomic probes corresponding to the PK/15 porcine circoviruses (PCV) and to the infectious chicken anaemia virus (CAV) were used. The plasmid pPCV1, containing the replicative form of the PCV genome, cloned in the form of a single 1.7 kilo base pair (kbp) insert (Meehan B. et al. Sequence of porcine circovirus DNA: affinities with plant circoviruses, J. Gen. Virol. 1997, 78, 221- 227), was used as specific viral DNA source for PCV. An analogous plasmid, pCAA1, containing the 2.3 kbp replicative form of the avian circovirus CAV was used as negative control. The respective glycerol stocks of the two plasmids were used for the production and purification of the plasmids according to the alkaline lysis technique (Sambrook J. et al. Molecular cloning: A Laboratory Manual. 2nd P:\OPER\jms\Specifcations76479-2002.10210 DIV.doc- 25/9/03 Edition, Cold Spring Harbor Laboratory, Cold Spring Harbor, 1989) so that they are then used as templates for the preparation of the probes. The circovirus Sprobes representative of the complete genomes of PCV and of CAV were produced from the purified plasmids described above (1 pg for each probe) and from hexanucleotide primers at random using a commercial nonradioactive 0 labelling kit ("DIG DNA labelling kit", Boehringer Mannheim, Lewes, UK) according 0- to the supplier's recommendations.

\0 The digoxigenin-labelled probes were taken up in a volume of 50-100 pl of sterile So0 water before being used for the in situ hybridization.

The diseased pig tissue samples, enclosed in paraffin and fixed with formaldehyde, as well as the preparations of infected cell cultures, fixed with formaldehyde, were prepared for the detection of the PCV nucleic acids according to the following technique: Sections 5 pm thick were cut from tissue blocks enclosed in paraffin, rendered paraffin free, and then rehydrated in successive solutions of alcohol in decreasing concentrations. The tissue sections and the cell cultures fixed with formaldehyde were incubated for 15 minutes and 5 minutes respectively at +37"C in a proteinase K solution in 0.05 M Tris-HCI buffer containing 5 mM EDTA (pH 7.6).

The slides were then placed in a 1% glycine solution in autoclaved distilled water, for 30 seconds, washed twice with 0.01 M PBS buffer (phosphate buffered saline) (pH and finally washed for 5 minutes in sterile distilled water. They were finally dried in the open air and placed in contact with the probes.

Each tissue/probe preparation was covered with a clean and fat-free glass coverslip, and then placed in an oven at +90 0 C for 10 minutes, and then placed in contact with an ice block for 1 minute, and finally incubated for 18 hours at +37 0

C.

The preparations were then briefly immersed in a 2 x sodium citrate salt (SSC) buffer (pH 7.0) in order to remove the protective glass coverslips, and then washed twice for 5 minutes in 2 x SSC buffer and finally washed twice for minutes in PBS buffer.

P: OPER\jrmSpciications764379.201302 120 DIV.doc- 239/03 SAfter these washes, the preparations were immersed in a solution of 0.1 M maleic acid, 0.15 M NaCI (pH 7.5) (maleic buffer) for 10 minutes, and then incubated in a 1% solution of blocking reagent (Cat #1096176, Boehringer Mannheim UK, Lewis, East Sussex, UK) in maleic buffer for 20 minutes at +37 0

C.

00 The preparations were then incubated with a 1/250 solution of an anti-digoxigenin 0 monoclonal antibody (Boehringer Mannheim), diluted in blocking buffer, for 1 hour N at +37"C, washed in PBS and finally incubated with a biotinylated anti-mouse 0 10 immunoglobulin antibody for 30 minutes at +37°C. The preparations were washed in PBS and the endogenous peroxidase activity was blocked by treatment with a hydrogen peroxide solution in PBS for 20 minutes at room temperature. The preparations were again washed in PBS and treated with a 3-amino-9diethylcarbazole (AEC) substrate (Cambridge Bioscience, Cambridge, UK) prepared immediately before use.

After a final wash with tap water, the preparations were counterstained with hematoxylin, "blued" under tap water, and mounted on microscope glass coverslips with a mounting fluid (GVA Mount, Cambridge Bioscience, Cambridge, UK) The experimental controls included the use of a nonpertinent negative probe (CAV) and of a positive probe (PCV) on samples obtained from diseased pigs and from nondiseased pigs.

Example A 4 Technique for the detection of PCV by immunofluorescence The initial screening of all the cell culture preparations fixed with acetone was carried out by an indirect immunofluorescence technique (IIF) using a 1/100 dilution of a pool of adult pig sera. This pool of sera comprises sera from 25 adult sows from Northern Ireland and is known to contain antibodies against a wide variety of porcine viruses, including PCV: porcine parvovirus, porcine adenovirus, and PRRS virus. The IIF technique was carried out by bringing the serum (diluted in PBS) into contact with the cell cultures for one hour at +37"C, followed by two washes in PBS. The cell cultures were then stained with a 1/80 dilution in PBS of a P;%OPEftjms\Spcciicaions7643792002302120 DIVdoc 259/03 rabbit anti-pig immunoglobulin antibody conjugated with fluorescein isothiocyanate for one hour, and then washed with PBS and mounted in glycerol buffer prior to Sthe microscopic observation under ultraviolet light.

Example A 5 Results of the in situ hybridization on diseased pig tissues 0 .0 The in situ hybridization, using a PCV genomic probe, prepared from tissues Scollected from French, Canadian and Califomian piglets having multisystemic Swasting lesions and fixed with formaldehyde, showed the presence of PCV nucleic 0 1o acids associated with the lesions, in several of the lesions studied. No signal was observed when the PCV genomic probe was used on tissues collected from nondiseased pigs or when the CAV probe was used on the diseased pig tissues.

The presence of PCV nucleic acid was identified in the cytoplasm and the nucleus of numerous mononuclear cells infiltrating the lesions in the lungs of the Californian piglets. The presence of PCV nucleic acid was also demonstrated in the pneumocytes, the bronchial and bronchiolar epithelial cells, and in the endothelial cells of the arterioles, the veinlets and lymphatic vessels.

In diseased French pigs, the presence of PCV nucleic acid was detected in the cytoplasm of numerous follicular lymphocytes and in the intrasinusoidal mononuclear cells of the lymph nodes. The PCV nucleic acid was also detected in occasional syncytia. Depending on these detection results, samples of Californian pig lungs, French pig mesenteric lymph nodes, and Canadian pig organs were selected for the purpose of isolating new porcine circovirus strains.

Example A 6 Results of the cell culture of the new porcine circovirus strains and detection by immunofluorescence No cytopathic effect (CPE) was observed in the cell cultures inoculated with the samples collected from French piglets (lmp.1008 strain), Californian piglets (lmp.999 strain) and Canadian piglets (lmp.1010 strain) showing clinical signs of multisystemic wasting syndrome. However, immunolabelling of the preparations obtained from the inoculated cell cultures, after fixing using acetone and with a P:\OPER\jms.Sp ciicaltons\764379-2002302120 DIV.doc. 25/9/03 Spool of pig polyclonal sera, revealed nuclear fluorescence in numerous cells in the cultures inoculated using the lungs of Californian piglets (lmp.999 strain), using the ;Z mediastinal lymph nodes of French piglets (Imp.1008 strain), and using organs of Canadian piglets (Imp. 1010 strain).

O Example A 7 Extraction of the genomic DNA of the porcine circoviruses (o o The replicative forms of the new strains of porcine circoviruses (PCV) were N prepared using infected PK/15 cell cultures (see Example A 1) (10 Falcons of cm 2 harvested after 72-76 hours of incubation and treated with glucosamine, as described for the cloning of the replicative form of CAV (Todd. D. et al. Dot blot hybridization assay for chicken anaemia agent using a cloned DNA probe. J. Clin.

Microbiol. 1991, 29, 933-939). The double-stranded DNA of these replicative forms was extracted according to a modification of the Hirt technique (Hirt B. Selective extraction of polyoma virus DNA from infected cell cultures, J. Mol. Biol. 1967, 36, 365-369), as described by Molitor (Molitor T. W. et al. Porcine parvovirus DNA: characterization of the genomic and replicative form DNA of two virus isolates, Virology, 1984, 137, 241-254).

Example A 8 Restriction map of the replicative form of the genome of the porcine circovirus Imp.999 strain.

The DNA (1-5 pg) extracted according to the Hirt technique was treated with SI nuclease (Amersh801 am) according to the supplier's recommendations, and then this DNA was digested with various restriction enzymes (Boehringer Mannheim, Lewis, East Sussex, UK) and the* products of digestion were separated by electrophoresis on a 1.5% agarose gel in the presence of ethidium bromide as described by Todd et al. (Purification and biochemical characterization of chicken anemia agent. J. Gen. Virol. 1990, 71, 819-823). The DNA extracted from the cultures of the lmp.999 strain possess a unique EcoRI site, 2 Sad sites and do not possess any Pstl site. This restriction profile is therefore different from the restriction profile shown by the PCV PK/15 strain (Meehan B. et al. Sequence of P:AOPER~jns\Sprciriio ionsk77437902002 02120 DIVdoc .5/103 porcine circovirus DNA; affinities with plant circoviruses, 1997 78, 221-227) which Spossess in contrast a Pstl site and do not possess any EcoRI site.

Example A 9 Cloning of the genome of the porcine circovirus lmp.999 strain 0 The restriction fragment of about 1.8 kbp generated by digestion of the double- 00 stranded replicative form of the PCV lmp.999 strain with the restriction enzyme SEcoRI was isolated after electrophoresis on a 1.5% agarose gel (see Example 3) 0 using a Qiagen commercial kit (QIAEXII Gel Extraction Kit, Cat #20021, QIAGEN So0 Ltd., Crawley, West Sussex, UK). This EcoRI-EcoRI restriction fragment was then ligated with the vector pGEM-7 (Promega, Medical Supply Company, Dublin, Ireland), previously digested with the same restriction enzymes and dephosphorylated, according to standard cloning techniques (Sambrook J. et al.

Molecular cloning: A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory, Cold Spring Harbor, 1989). The plasmids obtained were transformed into an Escherichia coli JM109 host strain (Stratagene, La Jolla, USA) according to standard techniques. The EcoRI-EcoRI restriction fragment of the PCV lmp.999 strain was also cloned into the EcoRI site of the vector pBlueScript SK+ (Stratagene Inc. La Jolla, USA) Among the clones obtained for each host strain, at least 2 clones containing the fragments of the expected size were selected. The clones obtained were then cultured and the plasmids containing the complete genome of the lmp.999 strain were purified in a small volume (2 ml) or in a large volume (250 ml) according to standard plasmid preparation and purification techniques.

Example A 10 Sequencing of a genomic DNA (doublestranded replicative form) of the PCV lmp.999 strain.

The nucleotide sequence of 2 EcoRI lmp.999 clones (clones pGEM-7/2 and pGEM-7/8) was determined according to Sanger's dideoxynucleotide technique using the sequencing kit "AmpliTaq DNA polymerase FS" (Cat #402079 PE Applied Biosystems, Warrington, UK) and an Applied BioSystems AB1373A automatic sequencing apparatus according to the supplier's recommendations.

P:'OPER'jnpcirFcaon7643792002302 120 DIV doc 25I9/03 SThe initial sequencing reactions were carried out with the M13 "forward" and S"reverse" universal primers. The following sequencing reactions were generated according to the "DNA walking" technique. The oligonucleotides necessary for these subsequent sequencings were synthesized by Life Technologies (Inchinnan Business Park, Paisley, UK).

00 1 The sequences generated were assembled and analysed by means of the SMacDNASIS version 3.2 software (Cat #22020101, Appligene, Durham, UK). The °various open reading frames were analysed by means of the BLAST algorithm to available on the "National Center for Biotechnology Information" (NCBI, Bethesda, MD, USA) server.

The complete sequence (EcoRI-EcoRI fragment) obtained initially from the clone pGEM-7/8 (SEQ ID No: 6) is presented in FIG. No. 6. It starts arbitrarily after the G of the EcoRI site and exhibits a few uncertainties from the point of view of the nucleotides.

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

The procedure was carried out in a similar manner for obtaining the sequence of the other three isolates according to the invention (see SEQ ID Nos: 1, 2 and 4 and FIGS. 1,2 and 4).

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

P:IOPCR~jjmllpcir~tions1764379-220023120 OIV.doc 2S19/03 SWhen the sequence generated from the Imp.999 strain was used to test for homology with respect to the sequences contained in the GenBank databank, the only significant homology which was detected is a homology of about 76% (at nucleic acid level) with the sequence of the PK/15 strain (accession numbers Y09921 and U49186) (see FIG. No. oo SAt amino acid level, the test for homology in the translation of the sequences in the N 6 phases with the databanks (BLAST X algorithm on the NCBI server) made it possible to demonstrate a 94% homology with the open reading frame corresponding to the theoretical replicase of the BBTV virus similar to the circoviruses of plants (GenBank identification number 1841515) encoded by the GenBank U49186 sequence.

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

Analysis of the sequences obtained from the lmp.999 strain cultured using lesions collected from Californian piglets having clinical signs of the multisystemic wasting syndrome shows clearly that this viral isolate is a new porcine circovirus strain.

Example A 12 Comparative analysis of the sequences The alignment of the nucleotide sequences of the 4 new PCV strains was made with the sequence of the PCV PK/15 strain (FIG. A homology matrix taking into account the four new strains and the previous PK/15 strain was established. The results are the following: 1 Imp. 1011-48121 2: Imp. 1011-48285 3: Imp. 999 4: Imp. 1010 P;lOPERjrm'Speciications764.379.2DO2302 120 DIVdoc. 25/9/03 1 2 3 4 1 1.000 0.9977 0.9615 0.9621 0.7600 2 1.000 0.9621 0.9632 0.7594 3 1.0000 0.9949 0.7560 4 1.0000 0.7566 5 1.0000 0 00

\O

1-

(N

0 The homology between the two French strains Imp. 1011-48121 and Imp. 1011- 48285 is greater than 99% (0.9977).

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

The homology between all these strains and PK/15 falls at a value between and 76%.

It is deduced therefrom that the strains according to the invention are representative of a new type of porcine circovirus, distinct from the type represented by the PK/15 strain. This new type, isolated from pigs exhibiting the PMWS syndrome, is called type II porcine circovirus, PK/15 representing type I.

The strains belonging to this type II exhibit remarkable nucleotide sequence *homogeneity, although they have in fact been isolated from very distant geographical regions.

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

The nucleotide sequence of the Imp. 1010 isolate was considered to be representative of the other circovirus strains associated with the multi-systemic wasting syndrome. This sequence was analysed in greater detail with the aid of the BLASTX algorithm (Altschul et al. J. Mol. Biol. 1990. 215. 403-410) and of a P:IOPERjmSpcica ionk7b43792002302120 DIV dec -2519/03 combination of programs from the set of MacVector 6.0 software (Oxford Molecular Group, Oxford OX4 4GA, UK) It was possible to detect 13 open reading frames (or ORFs) of a size greater than 20 amino acids on this sequence (circular genome). These 13 ORFs are the following: NAME START END STRAND Size of the Protein size ORF (amino acids (nucleotides (aa)) (nt)) ORF7 103 210 Sense 108 nt 35 aa ORF8 1180 1317 Sense 138 nt 45 aa ORF6 1363 1524 Sense 162 nt 53 aa ORF1 398 1342 Sense 945 nt 314 aa 900 1079 Sense 180 nt 59 aa ORF9 1254 1334 Sense 81 nt 26 aa ORF3 1018 704 Antisense 315 nt 104 aa 439 311 Antisense 129 nt 42 aa ORF11 190 101 Antisense 90 nt 29 aa ORF4 912 733 Antisense 180 nt 59 aa ORF12 645 565 Antisense 81 nt 26 aa ORF13 1100 1035 Antisense 66 nt 21 aa ORF2 314 1381 antisense 702 nt 233 aa The positions of the start and end of each ORF refer to the sequence presented in FIG. No. 4 (SEQ ID No. of the genome of strain 1010. The limits of ORFs 1 to 13 are identical for strain 999. They are also identical for strains 1011-48121 and 1011-48285, except for the ORFs 6 and 2: ORF6 1432-1539, sense, 108 nt, ORF2 314-1377, antisense, 705 nt, 234 aa.

Among these 13 ORFs, 4 have a significant homology with analogous ORFs situated on the genome of the cloned virus PCV PK-15. Each of the open reading frames present on the genome of all the circovirus isolates associated with the multisystemic wasting syndrome was analysed. These 4 ORFs are the following: PAOPER\jsm'SpciIiraions764379.2OO2302 120 DIVdoc. 19103

NO

0 0 oo

O

1

(N

!0 NAME START END STRAND Size of the Protein size Molecular ORF (nt) (aa) mass ORF1 398 1342 Sense 945 nt 314 aa 37.7 kDa ORF3 1018 704 Antisense 315 nt 104 aa 11.8 kDa ORF4 912 733 Antisense 180 nt 59 aa 6.5 kDa ORF2 314 1381 Antisense 702 nt 233 aa 27.8 kDa The positions of the start and end of each ORF refer to the sequence presented in FIG. No. 4 (SEQ ID No. The size of the ORF (in nucleotides=nt) includes the stop codon.

The comparison between the genomic organization of the PCV Imp. 1010 and PCV PK-15 isolates allowed the identification of 4 ORFs preserved in the genome of the two viruses. The table below presents the degrees of homology observed: 0 ORF Imp. 1010/ORF PVC PK-15 Percentage homology ORF1/ORF1 86 ORF2/ORF2 66.4 ORF3/ORF3 61.5 (at the level of the overlap (104 aa)) ORF4/ORF4 83 (at the level of the overlap (59 aa)) The greatest sequence identity was observed between ORF1 Imp. 1010 and ORF1 PK-15 (86% homology). This was expected since this protein is probably involved in the replication of the viral DNA and is essential for the viral replication (Meehan et al. J. Gen. Virol. 1997. 78. 221-227; Mankertz et al. J. Gen. Virol.

1998. 79. 381-384).

The sequence identity between ORF2 Imp. 1010 and ORF2 PK-15 is less strong (66.4% homology), but each of these two ORFs indeed exhibits a highly conserved N-terminal basic region which is identical to the N-terminal region of the major structural protein of the CAV avian circovirus (Meehan et al. Arch. Virol.

1992. 124. 301-319). Furthermore, large differences are observed between ORF3 Imp. 1010 and ORF3 PK-15 and between ORF4 Imp. 1010 and ORF4 PK-15. In each case, there is a deletion of the C-terminal region of the ORF3 and ORF4 of the Imp. 1010 isolate when they are compared with ORF3 and ORF4 of PCV PK- P:\OPERrjnmiSpcciications\764379.2002302120 DIVdoc 25/9/03 O 15. The greatest sequence homology is observed at the level of the N-terminal Sregions of ORF3/ORF3 (61.5% homology at the level of the overlap) and of SORF4/ORF4 (83% homology at the level of the overlap).

s It appears that the genomic organization of the porcine circovirus is quite complex as a consequence of the extreme compactness of its genome. The major structural protein is probably derived from splicing between several reading frames situated on the same strand of the porcine circovirus genome. It can therefore be Sconsidered that any open reading frame (ORF1 to ORF13) as described in the table above can represent all or part of an antigenic protein encoded by the type II porcine circovirus and is therefore potentially an antigen which can be used for specific diagnosis and/or for vaccination. The invention therefore relates to any protein comprising at least one of these ORFs. Preferably, the invention relates to a protein essentially consisting of ORF1, ORF2, ORF3 or ORF4.

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

The plasmid pGEM-7/8 containing the complete genome (replicative form) of the lmp.999 isolate was transfected into PK/15 cells according to the technique described by Meehan B. et al. (Characterization of viral DNAs from cells infected with chicken anemia agent: sequence analysis of the cloned replicative form and transfection capabilities of cloned genome fragments. Arch. Virol. 1992, 124, 301- 319). Immunofluorescence analysis (see Example 4) carried out on the first passage after transfection on noncontaminated PK/15 cells have shown that the plasmid of the clone pGEM7/8 was capable of inducing the production of infectious PCV virus. The availability of a clone containing an infectious PCV genetic material allows any useful manipulation on the viral genome in order to produce modified PCV viruses (either attenuated in pigs, or defective) which can be used for the production of attenuated or recombinant vaccines, or for the production of antigens for diagnostic kits.

P:SOPERtjn XSpeci cJicio-s'76437.200 2302I 20 DIV.d 2Y(9103 Example A 15 Production of PCV antigens by in vitro culture o0 The culture of the noncontaminated PK/15 cells and the viral multiplication were carried out according to the same methods as in Example A 1. The infected cells 0are harvested after trypsinization after 4 days of incubation at 37 0 C. and 0- enumerated. The next passage is inoculated with 400,000 infected cells per mi.

N Example A 16 Inactivation of the viral antigens 0 At the end of the viral culture, the infected cells are harvested and lysed using ultrasound (Branson Sonifier) or with the aid of a rotor-stator type colloid mill (UltraTurrax, IKA). The suspension is then centrifuged at 3700 g for 30 minutes.

The viral suspension is inactivated with 0.1% ethyleneimine for 18 hours at +37°C or with 0.5% beta-propiolactone for 24 hours at +28°C. If the virus titre before inactivation is inadequate, the viral suspension is concentrated by ultrafiltration using a membrane with a 300 kDa cut-off (Millipore PTMK300). The inactivated viral suspension is stored at +50 0

C.

Example A 17 Preparation of the vaccine in the form of an emulsion based on mineral oil.

The vaccine is prepared according to the following formula: suspension of inactivated porcine circovirus: 250 ml Montanide® ISA 70 (SEPPIC): 750 ml The aqueous phase and the oily phase are sterilized separately by filtration. The emulsion is prepared by mixing and homogenizing the ingredients with the aid of a Silverson turbine emulsifier.

One vaccine dose contains about 107.5 TCID50. The volume of one vaccine dose is 0.5 ml for administration by the intradermal route, and 2 ml for administration by the intramuscular route.

P:\OPER\jmstSpecificalions\764379.2002302120 DIV.doc- 25/9/03 Example A 18 Preparation of the vaccine in the form of a metabolizable oil-based emulsion.

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

One vaccine dose contains about 107.5 TCID50. The volume of one vaccine dose is 2 ml for administration by the intramuscular route.

Example A 19 The indirect immunofluorescence results in relation to the US and French PCV virus strains and to the PK/15 contaminant with a hyperimmune serum (PCV-T), a panel of monoclonal antibodies F99 prepared from PK/15 and a hyperimmune serum prepared from the Canadian strain (PCV-C)

VIRUS

USA France PCT-T antiserum 6400 200 800 PCV-C antiserum 200 6.400 2 6.400 F99 1H4 10 000 <100 100 F99 4B10 2 10 000 <100 <100 F99 2B7 10 000 100 <100 F99 2E12 10000 <100 <100 F991C9 10 000 <100 100 F99 2E1 <100 <100 F99 1H4 10 000 F99 1H4 100 <100 10000 P.\OPER\jms.Specrlcatiois76437-2002302120 DIV.dc- 25/9103 O Reciprocal of the last dilution of the serum or of the monoclonal antibody which Sgives a positive reaction in indirect immunofluorescence.

B. EXPERIMENTAL FOR ISOLATES 412, 9741 AND B9 MATERIALS AND METHODS 00 Cell Cultures. The Dulac cell line, a PCV-free PK15 derivative, was obtained from SDr. John Ellis (University of Saskatchewan, Saskatoon, Saskatchewan). The Vero 0cell line was obtained from American Type Culture Collection (ATCC), Manassas, VA. These cells were cultured in media suggested by the ATCC and incubated at 37*C with 5% CO 2 Porcine Circoviruses. The classic PCVI was isolated from persistently infected cells (ATCC CCL33). Isolate PCVII 412 was obtained from lymph nodes of a piglet challenged with the lymph node homogenate from PMWS-affected piglets. This challenged piglet had been diagnosed with PMWS. Isolate PCVII 9741 was isolated from the buffy-coat of peripheral blood from a PMWS-affected piglet of the same herd after the isolation of PCVII 412. Isolate PCVII B9 was isolated from an affected piglet in a United States swine herd with a PMWS clinical outbreak in the fall of 1997.

Propagation of PCVI. PCVI from persistently infected PK15 cells was grown and purified using a modified method of Tischer et al (1987) Arch. Virol. 96:39-57.

Briefly, PCV harvested from PK15 cells was used to super-infect a monolayer of PK15 cells at about 1 moi for two hours before the cells were treated with 300 mM D-glucosamine. After washing the cells once, DMEM (Gibco, catalog number 21013) with 5% FBS was added to the cells and the cells were incubated for an additional four days. The infected cells were scraped off and collected after centrifugation at 1500 x g for 15 minutes. The cell pellet was then treated with 0.5% of Triton X-114 at 37 0 C for 30 minutes. After another low speed centrifugation to remove cellular debris, an equal amount of Freon (Sigma catalog number T-5271) was added to the supernatant and the mixture was homogenized for one minute using a Polytron at maximum speed. The mixture was then P:IOPER'jms'Specircaruin 764379.200202 120 OIV.doc 2519103 centrifuged and the top layer collected and mixed with an equal volume of 0.1 M PBS. The virus pellet was collected after ultra centrifugation into a 20% sucrose Scushion at 210,000 x g for 30 minutes.

Culture of the Field Isolates (PCVII). The isolate PCVII 412 was cultured and purified in a similar manner as PCVI, except Dulac cells were used. The isolate 0 PCVII B9 was grown in heterogenic Vero cells transfected with self-ligated full- Slength PCR products from the United States PMWS outbreak. Therefore, the \0 possibility of contamination from other pig pathogens was eliminated. The B9transfected Vero cells were continuously passed and treated with 300 mM Dglucosamine as described above.

Viral DNA Isolation. Viral DNA was extracted from variable sources, including pellets of infected Dulac and Vero cells, peripheral blood buffy-coat cells, tissues from infected animals and serum. The tissue samples were treated with proteinase K and viral DNA was extracted using either phenol/chloroform or Qiagen tissue kit (Qiagen, Santa Clarita, Calif.). DNA from peripheral blood buffy coat cells of heparinized blood and serum was similarly collected using the Qiagen blood kit.

Infection of Piglets. Piglets were derived from specific pathogen-free sows. At one day of age, each piglet received approximately one gram of lymph nodes collected from PMWS-affected piglets. The tissue homogenate was distributed equally between the oral and intraperitoneal routes. Ten piglets were used in each of the experimental groups and observed daily for 7 weeks. Two groups were challenged and 2 were uninfected controls. Two groups, one challenged and one control, were also treated with cyclosporin A (2 mg/kg) at Day 0 and Day 14. The piglets were fed canned milk (Carnation) and water (50:50) until they self-weaned to high nutrient density commercially prepared feed.

PCR, Cloning and Sequencing of the Field PCV Isolates.

A two-step approach was used for the initial cloning of isolate PCVII 412 viral genomic DNA. A primer that hybridized to the conserved loop stem sequences, P: OPERjn-sSpccirKrtions 764379-200 2120 DIVdoc 291W03 O Loop' (Table was designed to perform a single-primed PCR taking advantage Sof the complementary sequences and the circular nature of PCV genomic DNA.

c-i The PCR reaction for the single-primed PCR was a two-stage process. The first stage consisted of 5 cycles of denaturing at 94°C for 1 minute, annealing at 37°C for 30 seconds and extension at 72 0 C for 2 minutes. The second stage consisted Sof 25 cycles of a similar program except the annealing temperature was increased to 52 0 C. The PCR products were cloned into a TA cloning vector (Invitrogen, Carlsbad, Calif.). Both strands of three different clones were sequenced to ensure Ssequence fidelity. Based on the sequences obtained, primer 1000- and R1F were designed in the noncoding region of the viral DNA sequences and used to clone the full-length viral genome. The sequences of all the primers used in this study are shown in Table 1. The sequences of the loop region were then obtained from the full-length clone. Sequences of isolate PCVII 9741 and PCVII 89 were obtained from purified PCR products. Automated DNA sequencing performed by Plant Biotechnology Institute of NRC, Canada was used with several internal primers. The sequences of isolates PCVII 412 (AF085695), PCVII 9741 (AF086835) and PCVII B9 (AF086834) have been deposited with the National Center for Biotechnology Information (NCBI).

Table 1 The sequences of Primers Used in the Studies Primer Name Primer Sequence SEQ ID NO: Loop' ACTACAGCAGCGCACTTC 19 1000- AAAAAAGACTCAGTAATTTATTTCATATGG R1F ATCACTTCGTAATGGTTTTTATT 21 1710+ TGCGGTAACGCCTCCTTG 22 850- CTACAGCTGGGACAGCAGTTG 23 1100+ CATACATGGTTACACGGATATTG 24 1570- CCGCACCTTCGGATATACTG 1230- TCCCGTTACTTCACACCCAA 28 400+ CCTGTCTACTGCTGTGAGTA 29 Sequence Analyses. The sequences of other circoviruses were obtained from NCBI. Various public domains were used for the sequence analysis, such as Biology workbench, Blast search, DNA/protein analysis tools, etc. The sequence P:OPER\jml\Spccifictions\7643.79-2002302120 DIV.doc 25/9/03 0 alignments were generated using Clustal W program (Biology Workbench, Sinternet address: http://biology.ncsa.uiuc.edu) and phylogenetic trees were created by PAUP 3.1 program (David L. Swofford, Laboratory of Molecular Systematics, MRC534, MRC at Smithsonian Institution, Washington, SMultiplex PCR. Two sets of primers were designed to identify the PCV group- 00 specific sequences and strain-specific sequences. The primer pair 1710+/850- is 0 PCV-group specific and 1100+/1570- is the novel PCV strain-specific pair, which differentiates the novel PCV from the one derived from PK15 cells. The two sets of primers have similar annealing temperatures for the PCR reaction and were used together at 0.5 .mu.M concentration in a standard hot start PCR reaction.

Either Ampli Taq Gold (Perkin Elmer) or Plentinum Taq (Gibco) was used.

Antiserum. The standard Berlin rabbit anti-PCVI antibody was kindly provided by Dr. Tischer (Koch Institute, Berlin, FRG). Rabbit anti-PCVII 412 pooled serum was obtained from two rabbits injected with purified isolate PCVII 412 at 50 pg/dose in an oil-in-water emulsion. The injection was repeated 3 times at 21-day intervals.

Pig anti-PMWS serum was collected from convalescent pigs from PMWS affected herds.

ELISA. Purified PCV was diluted in sodium carbonate buffer (0.05 M) pH 9.6 to a concentration of 0.5 pg per 100 pl and used to coat Immulon II plates (Dynatech Laboratories, Inc.). The plates were washed six times with TTBS (20 mM Tris- HCI, 500 mM NaCI, 0.05% of Tween 20, pH 7.5) before serially diluted primary rabbit or pig antibody was added. After six washes with TTBS, alkaline phosphatase-conjugated secondary antibodies (1/5000 dilution), either anti-rabbit or anti-pig (Kirkegaard Perry), were added. Plates were developed with 100 pl/well of p-Nitrophenyl Phosphate (PNPP, 3 g/L) in 1 M diethanolamine, MgCI 2 pH 9.8 and the plates were read on an ELISA reader (BioRad) at 405/490 nm.

FACS Analysis of Lymphocyte Surface Markers. Blood samples were collected from PMWS affected piglets in the field and negative control. The RBC was lysed P: OPER/jn/SpecilcaIions/764379.2002302 120 DIVdoc 25/9103 0 and WBC was stained with anti-pig CD3, CD4 and CD8 monoclonal antibodies, Sand followed by fluorescence labeled anti-mouse secondary antibody. The specifically labeled cells were fixed with 2% formaldehyde and 5000 cells were counted using FACS system (Becton Dickinson).

0 EXAMPLE B 1 00 oo 1 PMWS Reproduction PMWS has not been reproduced under controlled conditions, nor have etiology studies been performed. In order to determine the causative agent of this disease, a number of tissues were collected from PMWS-affected pigs, as described above in Materials and Methods, and studied. Lymph nodes displayed the most apparent gross lesions, histopathological changes and circovirus infection was confirmed by immunostaining. Accordingly, the lymph nodes were used in the challenge experiments described above.

The challenge experiments, conducted as described in Materials and Methods were successful in producing PMWS in pigs. In particular, some piglets died of the infection and asymptomatically infected piglets developed PMWS-like microscopic lesions by the end of the trial.

In another challenge experiment, the starting material used was lung tissue of pig with chronic wasting and lymph node enlargement. These clinical signs are characteristic of PMWS. The tissue was combined with sterile 0.1 M phosphatebuffered saline (PBS) and homogenized by passage through a polytron mixer.

The crude tissue homogenate was used to challenge pigs. In particular, a total of piglets (approximately 1 day of age) were randomly (balanced by litter of birth, gender and body weight) assigned to "tissue challenge," "tissue challenge with Cyclosporin-A," "control," or "Cyclosporin-A" treatment groups. The cyclosporin treatment had no clinical or hematological effect on the treated pigs except that cyclosporin was detected in the blood of those pigs three hours after the drug was administered. Hence, groups were collapsed across cyclosporin treatment for analysis.

P:%OPERljnamSpccilrions16439-2002302120 DIV.Ac. 2I9/03 SIn general, postmortem signs of PMWS disease in the challenged pigs included enlarged lymph nodes and incomplete collapse of lung tissue. Postmortem signs Sof PMWS disease were detected in significantly (p<0.01; two-tailed Fishers exacttest) more pigs in the group treated with tissue extract (7 pigs out of 9) than in the Sgroup treated with placebo (2 pigs out of 18). The average daily gain in the group treated by injection of tissue extract (212 gm/d) was not substantially different 0 from the group given the placebo (202 gm/d).

NO

Blood samples were obtained throughout the experiment and tissue samples were taken postmortem. The samples were tested for PCVII viral DNA by PCR, using PCR primers 1230- and 400+ (Table 1) which resulted in an 830 base pair product. Four of the pigs given the lung tissue extract had positive blood samples; whereas none of the pigs given placebo had PCVII DNA detected in their blood.

PCVII was detected in one or more tissues from 7 of the 8 surviving pigs in the "virus challenge" treatment group whereas all tissues from pigs in the control group were negative for PCVII. Contingency table analysis showed a significant difference (p<0.001; two-tailed Fishers exact-test).

In another challenge experiment, lung tissue of pig with chronic wasting and lymph node enlargement was collected and tissue debris removed by centrifugation (8000 rpm for 30 minutes). The supernatant was applied to a cesium chloride step-gradient and centrifuged at 100,00 x g. Bands appeared between 41% CsCI 2 (1.28 gm/ml) and 63% (1.40 gm/ml). These bands were applied to a 30% CsCI 2 "foot" and centrifuged for 2 hours at 100,000 x g. The pellet was resuspended in 15 mL of sterile 0.1 M PBS.

A total of 20 weaned piglets (approximately three weeks of age) were randomly (balanced by litter of birth, gender and body weight) assigned to "control" or "virus challenge" treatment groups. Pigs were weaned on Day 0 at approximately three weeks of age. In general, clinical signs of PMWS disease included enlarged lymph nodes and wasting or poor growth. Enlarged lymph nodes were detected in significantly (p<0.02; two-tailed Fisher exact-test) more pigs in the group treated P;%OPERjjnSpciricao 7641792002302120 DIVdoc- 219/03 O with virus (7 pigs) than in the group treated with placebo (1 pig). The average Sdaily gain in the group treated by virus injection (580 gm/d) tended to be less than the group given the placebo (616 gm/d), but the difference was not significant S(p=0.17; two-tailed paired t-Test). There was no difference between groups in the relative mass of internal organs (liver, lung, heart, spleen, kidneys).

00 oo Blood samples that were obtained throughout the experiment and tissue samples that were taken postmortem were tested for PCVII viral DNA using the PCR N techniques described above.

O All blood samples, including those taken just prior to euthanasia were negative for PCVII. PCVII was detected in one or more tissues for 8 of the 10 pigs in the "virus challenge" treatment group whereas all tested tissues from pigs in the control group were negative for PCVII. Contingency table analysis showed that this was a significant difference (p<0.001; two-tailed Fishers exact-test).

In conclusion, these experiments confirm that injection of weaned piglets with tissue extracts and gradient-purified viral material containing PCVII results in infection of multiple tissues. The infection persists for a duration of at least eight weeks.

EXAMPLE B -2 Isolation and Propagation of PCVII To determine the presence of an infectious causative agent(s) for PMWS, various tissues from pig #412, an experimentally challenged piglet sacrificed 21 days post-infection, were used for viral isolation. After continued passage of lymph node samples from pig #412 in Dulac cells, virus accumulation or adaptation was observed. A unique pattern of cytopathic effect initially developed, followed by increasing virus titer, as determined by ELISA using the standard Berlin anti-PCV antibody, as described above.

The existence of circovirus in Dulac cells infected with isolate PCVII 412 was P:IOPEAljrrks Speciriations%764M19.20 2120 DIV.doc 25/9/03 Sthen detected by electron microscopic examination. After six passages, viral structure proteins could be detected consistently, using a western blot assay.

EXAMPLE B 3 Specific Anti-PCVII Antibodies in Asymptomaticallv Infected and Convalescent 0 Piglets in PMWS-affected Herds 00 SBecause it appeared that porcine circoviruses possessed some heterogeneity, N ELISAs were performed using sera of piglets, collected from a herd with a PMWS outbreak, against the PCV and isolate PCVII 412 virus. Most of the asymptomatically PCVII-infected and convalescent piglets developed specific antibodies against PCVII, not PCVI.

EXAMPLE B -4 Isolation, Cloning and Sequencing of PCVII Virus and Viral Genomic DNA In order to explore genetic differences between the two strains of porcine circoviruses, viral DNA was extracted from infected Dulac cells. Considering the possible genetic unrelatedness between PCVI and PCVII, the approach was to design primer(s) from the most conserved region. Previous analysis of the PCV DNA sequences (Mankertz et al. (1997) J. Gen. Virol. 71:2562-2566; Meehan et al. (1997) J. Gen. Virol. 78:221-227) revealed a stem loop structure in the origin of replication. A single primer, targeting the inverted repeat sequence of the stem loop region, Loops, was designed because of the highly conserved nature of this important domain. The amplification of the PCVII 412 viral DNA by single primer PCR was successful. After cloning into a TA cloning vector, the viral genomic sequence was obtained by automated sequencing from several clones and both senses to ensure fidelity. The actual sequence of the stem loop or primer region was then obtained from a second full-length clone generated by primers of 1000- and RiF from the only non-coding region of the virus. The nucleotide sequence for PMWS 412 is shown in the top line of FIGS. 9A-9C.

Using similar primers, other PCVII isolates, including PCVII 9741 from the same P:\OPER\jnmuSpecirca:ions'76379002302I2O DIV.dc- 25/9/03 0 herd as PCVII 412, and PCVII B9 from a PMWS outbreak in the United States, Swere obtained. These strains were sequenced and compared to PCVII 412 and PCVI. See FIGS. 9A-9C for a comparison of PCVII 412 with PCVI and FIGS. 11A- 11B for comparisons of the PCVII 412 sequence with various PCV isolates.

The results of a phylogenetic analysis using the PAUP 3.1 program suggested 00 that the new PMWS isolates were closely related and in a different cluster with SPCVI. These isolates were therefore termed "PCVII" isolates. The percent Snucleotide sequence homologies among isolates of the novel porcine circovirus were more than 99% identical. In contrast, comparison of these nucleotide sequences with the PK15 PCVI showed only 75.8% overall nucleotide sequence homology. Comparative analysis of nucleotide sequences in different regions further revealed that the putative replication-associated protein gene of these two viruses share 81.4% homology, while the nucleotide sequences of the other large ORF was only 67.6% homologous.

Furthermore, nucleotide insertions and deletions (indels) were found in three regions. There are 13 base insertions in the new isolates between PCVI sequence 38-61 that flank the start codon for the putative 35.8 kd protein encoded by ORF 1. The area of PCVI 915-1033, containing 15 base indels, was at the ends and the joint region of the two largest ORFs (the other ORF was antisense) of the porcine circoviruses. The third region, covering PCVI sequence from 1529- 1735 with 15 base indels, locates at the amino end of a putative 27.8 kd protein encoded by ORF 2. PCVI sequences were also compared with the available sequences of the rest of the members of Circoviridae. PCVI is more closely related to banana bunch top virus (BBTV), a plant virus, than to chicken anemia virus (CAV) and beak and feather disease virus (BFDV) (both of which are avian circoviruses).

The gene map of isolate PCVII 412 is shown in FIG. 8. There are a total of six potential ORFs encoding proteins larger than 50 amino acid residues. A comparison between PCVII 412 and PK15 PCVI revealed homologies in four of the ORFs (Table The function of the 35.8 kd, namely the putative DNA P:\OPER\jrm\Specirictloni s64)79-M2002302120 DIV.doc 259/03

\O

O replicase protein, has been previously predicted (Meehan et al. (1997) J. Gen.

SVirol. 78:221-227). Analysis of these proteins predicted that both of the 35.8 kd and the antisense 27.8 kd proteins are nuclear proteins. Nucleotide sequence analysis also indicated that the start codons for the two proteins are within 33 bases of the origin of replication, which could also be the promoter. In addition, 0both ORFs ended with legitimate stop codons and poly A tail signals. Since some of the predicted proteins (based on size) could be found in western blots, these Sfindings suggest that porcine circoviral mRNA can be transcribed from both senses of the replicated forms. However, there is no coding sequence long enough to code for the common 31 kd protein and the additional 20 kd protein for the PCVII 412 isolate detected by western blot analysis. This suggests that posttranslational cleavage and/or RNA splicing may be involved in the expression of some of the porcine circovirus proteins.

Table 2 Putative Amino Acid Sequence Comparison Between PK15 PCVI and PCVII 412 Open reading frame Sequence Homology, Predicted Localization PCVI 412 PCVI/412 and Function 47-983 51-992 83.5 Nucleus, putative (ORF 1) (ORF 1) Rep protein 1723-1024 1735-1037 66.4 Nucleus (ORF 2) (ORF 2) 552-207 565-389 40.9 Endoplasmic Reticulum (ORF 4) (ORF 4) 658-40 671-359 29.1 Microbody (ORF 3) (ORF 3) EXAMPLE B Purification of PCVII Using Molecular Cloning Method Dulac cells were found to be infected with porcine retrovirus which is also found in many pig origin cell lines. In addition, other porcine pathogens were also found inconsistently associated with PCVII in PMWS-affected piglets. Thus, to obtain pure PCVII cultures, genetically cloned PCVII DNA was transferred to the P:\OPER\jrmlSpccifcaions\764379-2002302120 DIV.doc- 25/9/03 susceptible non-porcine origin Vero cells using liposomes. After two passages, Samplified PCV antigens were detected in the cells. The PCVII was seen to replicate and accumulate in the nuclei and was released into cytoplasm and other O cells during cell mitosis.

0EXAMPLE B 6 00 Multiplex PCR in PCVII Identification and PMWS Diagnosis 0 In order to differentiate the two strains of porcine circoviruses, PCVI and PCVII, 0 10 two sets of primers were designed based upon the comparative analysis of the viral DNA sequences. The PCV group-specific pair of 1710+/850, and isolate PCVII 412 strain-specific 1100+/1570-, were used in multiplex PCR for testing field samples. These primer sets were used with frozen tissues and buffy coat cells of peripheral blood. As judged by the multiplex PCR, using those primer sets, not only was PCVII infection identified in these samples but the genetic relatedness of the field samples was also determined. The presence of circovirus was later confirmed by electron microscopy.

The potency of this diagnostic method was further tested with another group of samples collected from a PMWS-affected herd (see FIG. 12). The PCVII DNA sequences could also be identified in almost all the tissues in PMWS-affected piglets (FIG. 13).

EXAMPLE B 7 PCVII Viremia Prior to and During PMWS Outbreak The development of PCR using serum enabled us to test the PCVII viremia in a swineherd showing specific anti-PCVII antibody. A group of 23 piglets was monitored from the age of one day until seven weeks and samples were collected at approximately two week intervals. A full-course of PCVII viremia and PMWS outbreak were observed, as indicated by the appearance to disappearance of the PCVII viremia which was detected in 9 of the 23 piglets. Most of piglets which showed PCVII viremia developed PMWS with some exhibiting severe PMWS.

P:\OPERyjmalSpcirlcJeions\76d37.20023o2 120 DIV.doc.- 299103 Table 3 shows the manifestation of PMWS in a typical pig. Gross lesions were found in most organs and tissues (Table 3).

TABLE 3 A clinical, histology, virology and immunology report of a typical PMWS affected piglet.

PMWS pig H254 Saliva Urine Bile Feces Serum Plasma Skin Fat Muscle Tongue Tonsil Cerv. LN Med. LN Mesenteric LN Inguinal LN Spleen Thymus Treachea Lung Heart Liver Gall Bladder Pancreas Adrenal Brain Eye Stomach Gross appearance Spine, hairy, disinterested and Wobbled

ND

Pale/clear Thin, not viscid Scant but normal

N

Yellow Hint of yellow Little/no fat Histopath PCR

N

Small crypts Enlarged Very large, dark surface, yellow center Very enlarged, dark and wet Large, dark and wet Small and thin Small and difficult to find

N

A, M lobes 80% atelectasis; firm texture mottles and spots thoughout all lobes Thin and flabby "Camouflage" pattem mottling N, moderately full Glossitis Lymphocyte depletion Lymphocyte depletion Lymphocyte depletion Lymphocyte depletion Lymphocyte depletion Lymphocyte depletion

ND

Metaplasia adenitis Interstitial Pneumonia Focal adrenalitis Meningitis N, white sclera N, full of feed P:OPER'jffspcirctions76I79202302 1 20 DIV.ac 2519/03 Small intestine Large intestine Kidney Urinary bladder

CBC

FACS

N

N, sandy/gritty contents Enlarged, dark and no pus WBC: 20.1 Segs: 62% or 12.462 Lymphs: 29.0% or 5.829 CD3: 52.1% CD4: 9.0% CD8: 66.5% Peyers Patch Submucosal inflam Interstitial nephritis Ref mg X 11.0-22.0 3.08-10 4.29-13.6 EXAMPLE B 8 Host Immune System Dysfunction in PMWS Affected Piglets It is interesting that while lymphocyte infiltration was discovered in most of the tissues, lymphocyte depletion was consistently found in all the lymphoid tissues (Table Decreased CD4 cell, and increased CD8 cells were also seen, while CD3 cells remained relatively stable (Table 4, mean numbers are from two PMWS affected and 40 negative control piglets). These changes resulted in CD4/CD8 ratio which drastically dropped from 1.58 to 0.13. These finding suggested that PCVII could induce host immune system malfunction and therefore suppress the host immune responses to PCVII and possibly other pathogens. Thus, PMWS appears to be a disease of immunodeficiency in piglets.

PM

Cor Table 4 Lymphocyte Surface Markers of PMWS Affected and Control of 6-week-old Piglets CD3 CD4 CD8 C04/CD8 ratio WS 59.88 8.85 67.6 0.13 Itrol 53.46 24.02 15.18 1.58 Thus, the cloning, expression and characterization of novel PCVII isolates is disclosed, as are methods of using the same. Although preferred embodiments of the subject invention have been described in some detail, it is understood that obvious variations can be made without departing from the spirit and the scope of the invention as defined by the appended claims.

P.%OPERjrrSpecirionsN764379-.20232O220 DIVdoc. 2519/03 Throughout this specification and the claims which follow, unless the context requires otherwise, the word "comprise", and or variations such as S"comprises" or "comprising", will be understood to imply the inclusion of a stated C integer or step or group of integers or steps but not the exclusion of any other 0 integer or step or group of integers or steps.

0 o00 7- The reference to any prior art in this specification is not, and should not be 0 taken as, an acknowledgment or any form of suggestion that that prior art forms IN0 part of the common general knowledge in Australia.

1 0 P:NOPER jr I0Spcircclions\6437920020212 DIV.doc 25/903

Claims (23)

1. An isolated nucleotide sequence encoding porcine circovirus type II (PCVII), Scapable of being isolated from a physiological sample or from a tissue sample o from a diseased pig having post-weaning multisystemic wasting syndrome (PMWS). 0 S2. The isolated nucleotide sequence according to claim 1, wherein the nucleotide 0 sequence is isolated from a lesion. O IND

3. A cell line infected with the isolated nucleotide sequence according to claim 1.

4. The cell line according to claim 1, which is a PK/15 cell line. An isolated nucleotide sequence encoding PCVII, wherein the nucleotide sequence is isolated from the cell line according to claim 3, or from a culture extract or supernatant thereof.

6. A vaccine against PMWS, wherein the vaccine comprises a PCVII antigen.

7. The vaccine according to claim 6, wherein the PCVII antigen is an attenuated live whole PCVII virus.

8. The vaccine according to claim 7, further comprising a veterinarily acceptable vehicle, diluent, adjuvant and/or freeze-drying stabilizer.

9. The vaccine according to claim 8, wherein the freeze-drying stabilizer is selected from the group consisting of SPGA, sorbitol, mannitol, starch, sucrose, dextran, glucose, albumin, casein and alkali metal phosphates.

10. The vaccine according to claim 7, comprising 103 to 106

11. The vaccine according to claim 6, wherein the PCVII antigen is an inactivated PCVII virus.

12. The vaccine according to claim 11, further comprising a veterinarily acceptable vehicle, diluent, and/or adjuvant. m:\specifications\500000\504000\54406clmmjc.doc S13. The vaccine according to claim 11, comprising 10 6 to 108 S14. The vaccine according to claim 8 or 12, provided in the form of a water-in-oil or oil-in-water emulsion. O 00 The vaccine according to claim 8 or 12, wherein the adjuvant is selected from O the group consisting of aluminium hydroxide, saponin, avridine, \O dimethyldioctadecylammonium bromide, polyphosphazene and combinations thereof.

16. The vaccine according to claim 13, wherein the adjuvant is selected from the group consisting of mineral oil or other metabolizable oil, squalane, squalene, and combinations thereof.

17. An isolated nucleotide sequence encoding a PCVII polypeptide selected from the group consisting of a polypeptide derived from ORF 1, ORF 2, ORF 3, ORF 4, ORF 5, ORF 6, and an immunogenic fragment thereof comprising at least amino acids.

18. A polypeptide expressed from the nucleotide sequence according to claim 17.

19. A vaccine comprising the polypeptide according to claim 18.

20. The isolated nucleotide sequence according to claim 17, wherein the nucleotide sequence is placed under the control of a promoter.

21. An in vitro or in vivo expression vector comprising the nucleotide sequence according to claim

22. The in vivo expression vector according to claim 21, wherein the vector is selected from the group consisting of poxviruses, adenoviruses, herpesviruses and DNA vectors.

23. A composition comprising the in vivo expression vector according to claim 21 and a veterinarily acceptable vehicle. m:\specifications\500000\504000\5044O6clmmjc.doc S24. The in vitro expression vector according to claim 21, wherein the vector is baculovirus. O

25. A host cell transformed with the in vitro expression vector according to claim 0 22. 00 S26. The host cell according to claim 25, which is an E. coli cell. (O

27. A method for producing a recombinant PCVII polypeptide comprising providing a population of host cells according to claim 25 or 26, and culturing the host cells under conditions whereby the PCVII polypeptide encoded by the nucleotide sequence is expressed.

28. A polypeptide produced by the method of claim 27.

29. A composition comprising the polypeptide according to claim 28, a veterinarily acceptable vehicle and, optionally, an adjuvant.

30. The composition according to claim 29, wherein the adjuvant is selected from the group consisting of muramyl dipeptide, avridine, aluminium hydroxide, dimethyloctadecyl ammonium bromide, oil, oil-in-water emulsion, saponin and cytokine. Dated this ninth day of June 2006 Merial; The Queen's University of Belfast; University of Saskatchewan Patent Attorneys for the Applicant: F B RICE CO m:\specifications\500000\504000\5044O6clmmjcdoc