BEST MODE FOR CARRYING OUT THE INVENTION

[0015] The present invention relates to compositions and methods for culturing mammalian circovirus, in particular porcine circovirus. The present invention is based on the finding that a porcine cell expressing human E1 function was able to be transfected with a PCV2 virus genome and generated PCV2 virus with a high virus titer. VIDO R1 cell line, deposited with the ATCC and having ATCC accession number PTA-155, is a porcine retina cell line transformed with human adenovirus-5 (HAV5) E1, that has been shown to induce the S phase of the cell cycle and transactivate transcription. See, Shenk, T. (1996). “ Adenoviridae : the viruses and their replication” In Fields Virolog. 3 ^rd ed. B. N. Fields, D. M. Knipe and P. M. Howley (ed.) Lippincott-Raven Publishers, Philadelphia, N.Y., pp. 2111-2148. As described herein in Example 3, VIDO R1 cells, were transfected with a PCV2 genome and generated virus at 2×10 ⁷ IU/ml.

[0016] The practice of the present invention employs, unless otherwise indicated, conventional microbiology, immunology, virology, molecular biology, and recombinant DNA techniques which are within the skill of the art. These techniques are fully explained in the literature. See, e.g., Maniatis et al., Molecular Cloning: A Laboratory Manual (1982); DNA Cloning: A Practical Approach , vols. I & II (D. Glover, ed.); Oligonucleotide Synthesis (N. Gait, ed. (1984)); Nucleic Acid Hybridization (B. Hames & S. Higgins, eds. (1985)); Transcription and Translation (B. Hames & S. Higgins, eds. (1984)); Animal Cell Culture (R. Freshney, ed. (1986)); Perbal, A Practical Guide to Molecular Cloning (1984); Ausubel, et al., Current Protocols In Molecular Biology , John Wiley & Sons (1987, 1988, 1989, 1990, 1991, 1992, 1993, 1994, 1995, 1996); and Sambrook et al., Molecular Cloning: A Laboratory Manual (2 ^nd Edition); vols. I, II & III (1989).

[0017] Circoviridae, a family of viruses having round, non-enveloped virions with mean diameters from 17 to 23.5 nm containing circular, single-stranded DNA (ssDNA), are described in The Sixth Report of the International Committee for the Taxonomy of Viruses, supra. Members of the group include the porcine circoviruses, PCV1 and PCV2. Some of the PCVs are known to be pathogenic, such as PCV2, associated with PMWS.

[0018] Nucleotide sequences for PCV1 are provided in Mankertz, A., et al., 1997 , J. Virol. 71:2562-2566 and Meehan, B. M. et al., 1997 , J. Gen. Virol. 78:221-227. Nucleotide sequences for PCV2 are provided in Hamel, A. L. et al., 1998 , J. Virol. 72:5262-5267; Mankertz, A. et al., 2000, Virus Res. 66:65-77 and Meehan, B. M. et al., 1998 , J. Gen. Virol. 79:2171-2179. Representative strains of PCV2 have been deposited with the European Collection of Cell Cultures, Centre for Applied Microbiology & Research, Porton Down, Salisbury, Wiltshire SP4 OJG, United Kingdom and include accession No. V97100219; accession No. V9700218; accession No. V97100217; accession No. V98011608; and accession No. V98011609. WO 00/77216 also discloses PCV2. PCV2 nucleotide sequences have also been published in Hamel et al., (1998), J. Virol. vol. 72, 6:5262-5267 (GenBank AF027217) and in Morozov et al., (1998), J. Clinical Microb. vol. 36, 9:2535-2541, as well as GenBank AF086834; AF086835; and AF086836. Comparison of the published nucleotide sequences for PCV1 and PCV2 reveals a <80% identity, although the genomic organization is similar, especially in the arrangement of the two largest open reading frames (ORFs) with a putative origin of DNA replication.

[0019] The present invention encompasses methods of culturing mammalian circovirus and in particular, porcine circovirus (PCV). The present invention encompasses methods of culturing PCV comprising the PCV nucleotide sequences disclosed herein or known in the art, or ORFs thereof, or portions thereof that are capable of replication. The present invention also encompasses methods of culturing PCV having PCV nucleotide sequences differing through the degeneracy of the genetic code to those disclosed herein or known in the art, or ORFs thereof, or portions thereof capable of replication. The present invention further encompasses methods of culturing PCV comprising PCV nucleotide sequence variations which do not change the functionality or strain specificity of the nucleotide sequence, or ORFs thereof, or portions thereof capable of replication. The present invention also encompasses methods of culturing PCV comprising PCV nucleotide sequences capable of hybridizing to those sequences disclosed herein under conditions of intermediate to high stringency, and methods of culturing PCV comprising mutations of the PCV nucleotide sequence disclosed herein or known in the art, such as deletions or point mutations, or ORFs thereof, or portions thereof, capable of replication. The present invention also encompasses methods of culturing PCV comprising heterologous nucleotide sequences. The present invention encompasses methods of culturing PCV that comprise chimeric circovirus nucleotide sequences, such as, for example, nucleotide sequences from porcine circovirus in fusion with nucleotide sequences from other pathogenic viruses, such as a pathogenic porcine virus, including parvovirus.

[0020] As used herein, a heterologous nucleotide sequence, with respect to a circovirus or mammalian cell, is one which is not normally associated with the circovirus sequences as part of the circovirus genome or one which is not normally associated with the mammalian cell, respectively. Heterologous nucleotide sequences include synthetic sequences. Hybridization reactions can be performed under conditions of different “stringency”. Conditions that increase stringency of a hybridization reaction are widely known and published in the art. See, for example, Sambrook et al. (1989) at page 7.52. Examples of relevant conditions include (in order of increasing stringency): incubation temperatures of 25° C., 37° C., 50° C. and 68° C.; buffer concentrations of 10× SSC, 6× SSC, 1× SSC, 0.1× SSC (where SSC is 0.15 M NaCl and 15 mM citrate buffer) and their equivalents using other buffer systems; formamide concentrations of 0%, 25%, 50%, and 75%; incubation times from 5 minutes to 24 hours; 1, 2, or more washing steps; wash incubation times of 1, 2, or 15 minutes; and wash solutions of 6× SSC, 1× SSC, 0.1× SSC, or deionized water. An exemplary set of stringent hybridization conditions is 68° C. and 0.1× SSC.

[0021] The PCV genomes encode several polypeptide sequences, ranging in approximate size from 8 to 35 kD. It is deemed routine to determine open reading frames (ORFs) for porcine circoviruses using standard software such as for example, MacVector® (Oxford Molecular Group Inc., MD 21030). The largest ORF, ORF1, of the two types of PCV shows only minor variation with an identity of 85% (as measured by the clustal program) and has been demonstrated to be the Rep protein in PCV1 (Mankertz, A., et al., 1998 , J. Gen. Virol. 79:381-384). Without wanting to be bound by theory, a higher rate of variation displayed in the ORF2 sequences of PCV1 and PCV2 (identity about 65%) would suggest that type-specific features of PCV might be determined by the respective ORF2 protein. Several PCV type-specific epitopes have been mapped on PCV2 ORF2 sequences. See Mahe, D. et al., 2000 , J. Gen. Virol. 81:1815-24. In another recent study, PCV2 ORF2 has been identified as a major structural protein that can form viral capsid-like particles in insect cells infected with ORF2 expressing recombinant baculovirus. See Nawagitgul, P. et al., 2000 , J. Gen. Virol. 81:2281-2287.

[0022] In some illustrative embodiments of the invention disclosed herein, a recombinant vector comprising a PCV genome or an ORF thereof, or a portion thereof, such as an antigenic region, is constructed by in vitro recombination between a plasmid and a PCV genome. In some embodiments, the PCV genome is a PCV2 genome. In other embodiments, a recombinant vector comprising a PCV genome or an ORF thereof, or a portion thereof, such as an antigenic region, is constructed by in vivo recombination. Methods for in vivo recombination are know in the art and include, for example, the methods disclosed in Chartier, et al. (1996 , J. Virol. 70:4805-4810). Vectors for constructing circovirus genomes include for example, bacterial plasmids which allow multiple copies of the cloned circovirus nucleotide sequence to be produced. In some embodiments, the plasmid is co-transfected into a suitable host cell for recombination. Suitable host cells for recombination include any cell that will support recombination between a PCV genome and a plasmid containing PCV sequences, or between two or more plasmids, each containing PCV sequences. Recombination is generally performed in procaryotic cells, such as E. coli for example, while generation of circovirus is preferably performed in mammalian cells permissive for PCV replication, such as for example porcine cells and in particular, porcine cells capable of expressing mammalian adenovirus E1 function.

[0023] The present invention encompasses the use of any mammalian host cell permissive for circovirus replication, and in particular, permissive for replication of PCV, such as PCV1 and PCV2. Allan et al. (1995 , Veterinary Microbiology 44: 49-64) report that PCV replicate in porcine and bovine monocyte/macrophage cultures. Tischer et al. (1987 , Arch. Virol. 96:39-57) report that PCV is known to require actively dividing cells for replication in cell culture. Examples of cells or cell lines useful for replication of PCV include mammalian cells comprising E1 function and permissive for PCV replication, including porcine cells, such as porcine monocyte/macrophage cells and porcine retinal cells, expressing adenovirus E1 function. In an illustrative embodiment disclosed herein, porcine retina cells expressing human adenovirus E1 function are shown to be permissive for replication of PCV2 and shown to generate virus at 2×10 ⁷ IU/ml. Porcine cell lines are available from public sources such as for example, the American Type Tissue Collection (ATCC). The growth of bacterial cell cultures, as well as culture and maintenance of eukaryotic cells and mammalian cell lines are procedures well-known to those of skill in the art.

[0024] The present invention encompasses methods of culturing mammalian circovirus, in particular, porcine circovirus, in mammalian host cells transfected with mammalian adenovirus E1 gene sequences. In some embodiments, the mammalian cell is stably transformed with adenovirus E1 gene sequences. In some embodiments, the E1 gene sequences are integrated into the genome of the mammalian cell. In other embodiments, the E1 gene sequences are present on a replicating plasmid. In yet other embodiments, the E1 gene sequence is heterologous to the mammalian cell. In an illustrative embodiment disclosed herein a porcine mammalian cell is transformed with a human adenovirus 5 E1 gene sequence. The present invention encompasses the use of any mammalian cell or mammalian cell line expressing E1 function as long as the mammalian cell or cell line expressing E1 function is permissive for the replication of circovirus, in particular porcine circovirus, such as for example, porcine circovirus 1 or porcine circovirus 2. In preferred embodiments, the mammalian cell is a porcine cell or cell line. The present invention encompasses the use of any mammalian E1 function as long as the mammalian host cell expressing the mammalian E1 function is permissive for replication of circovirus, in particular, PCV, such as for example, porcine circovirus 1 or porcine circovirus 2. Mammalian adenovirus genomes are known in the art and are disclosed in, for example, Reddy et al. (1998 , Journal of Virology, 72:1394) which discloses nucleotide sequence, genome organization, and transcription map of bovine adenovirus 3 (BAV3); and Kleiboeker (1995 , Virus Res. 36:259-268), which discloses the E1 region of PAV4. The present invention encompasses E1 function from any of the various serotypes of human adenovirus, such as Ad2, Ad5, Ad12, and Ad40. In an illustrative embodiment disclosed herein in Example 1, E1 function is human Ad5 E1 function. The human E1A gene is expressed immediately after viral infection (0-2 hours) and before any other viral genes. Flint (1982) Biochem. Biophys. Acta 651:175-208; Flint (1986) Advances Virus Research 31:169-228; Grand (1987) Biochem. J. 241:25-38. The transcription start site of Ad5 E1A is at nucleotide 498 and the ATG start site of the E1A protein is at nucleotide 560 in the virus genome. The E1B protein functions in trans and is necessary for transport of late mRNA from the nucleus to the cytoplasm. The E1B promoter of Ad5 consists of a single high-affinity recognition site for Sp1 and a TATA box. In particular, human adenovirus 5 E1A and E1B gene sequences are located at nucleotides 5054034 of the nucleotide sequence provided in Chroboezek, J. et al. (1992 , Virology. 186:280-285). In an illustrative embodiment disclosed herein in the Examples, the mammalian host cell is a porcine host cell transfected with human adenovirus 5 E1 gene sequences.

[0025] The PCV genome can be isolated from PCV virions, or can comprise a PCV genome that has been inserted into a plasmid, using standard techniques of molecular biology and biotechnology. Cloning of the full-length PCV2 genome into vector pBluescript II KS(+) from Strategene by PCR is described in Liu, et al. (2000 , J. Clin. Microbiol. vol 38:3474-3477). The full-length PCV2 genome DNA can be released from the resulting plasmid upon SacII digestion.

[0026] Introduction of circovirus nucleotide sequences into permissive mammalian host cells can be achieved by any method known in the art, including, but not limited to, transfection and transformation including, but not limited to, microinjection, electroporation, CaPO ₄ precipitation, DEAE-dextran, liposomes, particle bombardment, etc. An illustrative method for transfecting PCV2 nucleotide sequences into VIDO R1 cells is described herein in Example 3.

[0027] Methods for culturing procaryotic cells, such as bacterial cells, and eukaryotic cells, such as mammalian host cells expressing adenovirus E1 function are deemed routine to those of skill in the art.

[0028] The following examples are provided to illustrate but not limit the invention. All references and patent publications disclosed herein are hereby incorporated in their entirety by reference.

EXAMPLES

Example 1

Preparation of Porcine Retinal Cells Transfected with Human Adenovirus E1 Gene Sequences (VIDO R1 cells)

[0029] Primary cultures of porcine embryonic retina cells were transfected with 10 μg of plasmid pTG 4671 (Transgene, Strasbourg, France) by the calcium phosphate technique. The pTG 4671 plasmid contains the entire E1A and E1B sequences (nts 505-4034) of HAV-5, along with the puromycin acetyltransferase gene as a selectable marker. In this plasmid, the E1 region is under the control of the constitutive promoter from the mouse phosphoglycerate kinase gene, and the puromycin acetyltransferase gene is controlled by the constitutive SV40 early promoter. Transformed cells were selected by three passages in medium containing 7 μg/ml puromycin, identified based on change in their morphology from single foci (i.e., loss of contact inhibition), and subjected to single cell cloning. The established cell line was first tested for its ability to support the growth of E1 deletion mutants of HAV-5. Subsequently the cell line was further investigated for the presence of E1 sequences in the genome by PCR, expression of the E1A and E1B proteins by Western blot, and doubling time under cell culture conditions. E1 sequences were detected, and production of E1A and E1B proteins was demonstrated by immunoprecipitation. Doubling time was shorter, when compared to that of the parent cell line.

[0030] To assess the stability of E1 expression, VIDO R1 cells were cultured through more than 50 passages (split 1:3 twice weekly) and tested for their ability to support the replication of E1-deleted HAV-5. Expression of the E1A and E1B proteins at regular intervals was also monitored by Western blot. The results indicated that the VIDO R1 line retained the ability to support the growth of E1-deleted virus and expressed similar levels of E1 proteins during more than 50 passages in culture. Therefore, VIDO R1 can be considered to be an established cell line. VIDO R1 cell line has been deposited with the American Type Culture collection (ATCC) and has ATCC accession number PTA-155.

Example 2

[0031] Example 2 provides a description of the molecular cloning of full-length PCV2 genome.

[0032] Initially, PCV2 DNA was amplified by PCR from total DNA extracted from a piglet with PMWS. The cloning of the full-length PCV2 genome DNA into vector pBluescript II KS(+) (Stratagene) by polymerase chain reaction (PCR) was described in Liu et al. (2000). J. Clin. Microbiol. 38:3474-3477). The PCV2 sequence was submitted to GenBank (Accession no. AF086834). The full-length PCV2 genome DNA is released from the resulting plasmid upon SacII digestion.

Example 3

[0033] Example 3 describes the transfection of VIDO R1 cells, as described in Example 1, with a plasmid containing the PCV2 genome as constructed in Example 2.

[0034] Material and Methods

[0035] Cell Culture

[0036] Fetal porcine retina cell line, VIDO R1, as described in Example 1 and Vero cells (ATCC) were maintained at 37° C. with 5% CO ₂ in Eagles based MEM media supplemented with 10% or 5% heat-inactivated fetal bovine serum (FBS), respectively.

[0037] Transfection and Infection

[0038] Monolayers of VIDO R1 cells grown in a six-well dish were transfected with cloned PCV2 DNA using Lipofectin according to the manufacturer's recommendations (GIBCO BRL). Prior to transfection, PCV2 full-length genome was released from the plasmid by digestion with SacII (Liu, Q., et al., 2000 , J. Clin. Microbiol. 38:3474-3477). For infection, the transfected VIDO R1 cells were subjected to three cycles of freezing (−70° C.) and thawing (37° C.). The lysate was then clarified by centrifugation and used to infect fresh VIDO R1 cells. In published reports, a PCV 1-free porcine kidney cell line is used to culture PCV2 virus. To stimulate the entry to the S phase in the cell cycle, the porcine kidney cells are always treated by D-glucosamine (see Tischer et al., (1987). Arch Virol. 96:39-57.). However, the treatment must be performed with caution because D-glucosamine is toxic for cell culture (see, Allan et al., (2000). J. Vet. Diagn. Investigation. 12:3-14). In contrast, since the VIDO R1 cell line used in this study has been transformed by HAV5-E1 that can induce the S phase, the D-glucosamine treatment was not necessary.

Example 4

[0039] Virus Purification and Titration

[0040] For the purification of PCV2 virus, PCV2-infected VIDO R1 cells were incubated with 0.5% Triton X-114 in phosphate-buffered saline (PBS) at 37° C. for 45 min followed by Freon 113 (1,1,2-trichloro-trifluoroethane) extraction. The cell debris and membranes were clarified by centrifugation at 2000 g for 15 min. The viruses in the supernatant were pelleted at 35000 g for 3 h through a 20% sucrose cushion. The virus pellet was suspended in PBS and stored at −70° C. Virus titers were determined as infectious units (IU) by quantitative ORF2 protein immuno-peroxidase staining. For this purpose, the cell monolayers in 12-well dishes were infected with serial dilutions of virus. After adsorption of virus for 1 h, the cells were washed and overlaid with MEM containing 2% FBS and 0.7% agarose. On day 3 post infection (p.i.), the agarose overlay was removed and the cells were fixed and permeabilized with methanol/acetone (1:1 in volume) for 20 min at −20° C. After blocking with 1% bovine serum albumin for 1 h at room temperature, the cells were incubated with rabbit anti-ORF2 serum (Liu et al., 2001, Protein Expression and Purification. 21:115-120). After 2 h incubation, the plates were washed with PBS and then processed using VECTASTAIN Elite ABC kit (Vector Laboratories). The reaction was developed with 3,3′diaminobenzidine (DAB) tetrahydrochloride and observed under a microscope. By counting the positively stained cells, the virus titer was expressed as IU where 1 IU was defined as one positively stained cell/foci at 3 d.p.i.

[0041] Viral DNA Extraction and Characterization

[0042] Viral DNA was extracted from PCV2-infected VIDO R1 cell monolayers by the method of Hirt (1967 , J. Mol. Biol. 26:365-369). The viral DNA was then characterized by restriction analysis and polymerase chain reaction (PCR) as described in Liu et al., (2000). J. Clin. Microbiol. 38:3474-3477).

[0043] PCR using DNA extracted from the infected cells as template and PCV-2-specific primers amplified a product of specific size, while no DNA was amplified from control, uninfected cells. Consistent with the expected restriction patterns, digestions of viral DNA with NcoI and StuI resulted in two fragments of 1291 bp and 477 bp in size, respectively; digestion with EcoRI and StuI produced two fragments of 1492 bp and 276 bp in size, respectively; and digestion with EcoRI and EcoRV generated two fragments of 1094 bp and 674 bp in size, respectively. The data indicate that PCV2 virus was obtained. Using an immunostaining assay and by counting the positive stained cells, the virus titer of this preparation was determined to be 2×10 ⁷ IU/ml.