PMCCPMCCPMCC

Search tips
Search criteria 

Advanced

 
Logo of jvirolPermissionsJournals.ASM.orgJournalJV ArticleJournal InfoAuthorsReviewers
 
J Virol. 2010 September; 84(17): 8986–8989.
Published online 2010 June 23. doi:  10.1128/JVI.00522-10
PMCID: PMC2919042

Replacement of the Replication Factors of Porcine Circovirus (PCV) Type 2 with Those of PCV Type 1 Greatly Enhances Viral Replication In Vitro[down-pointing small open triangle]

Abstract

Porcine circovirus type 1 (PCV1), originally isolated as a contaminant of PK-15 cells, is nonpathogenic, whereas porcine circovirus type 2 (PCV2) causes an economically important disease in pigs. To determine the factors affecting virus replication, we constructed chimeric viruses by swapping open reading frame 1 (ORF1) (rep) or the origin of replication (Ori) between PCV1 and PCV2 and compared the replication efficiencies of the chimeric viruses in PK-15 cells. The results showed that the replication factors of PCV1 and PCV2 are fully exchangeable and, most importantly, that both the Ori and rep of PCV1 enhance the virus replication efficiencies of the chimeric viruses with the PCV2 backbone.

Porcine circovirus (PCV) is a single-stranded DNA virus in the family Circoviridae (34). Type 1 PCV (PCV1) was discovered in 1974 as a contaminant of porcine kidney cell line PK-15 and is nonpathogenic in pigs (31-33). Type 2 PCV (PCV2) was discovered in piglets with postweaning multisystemic wasting syndrome (PMWS) in the mid-1990s and causes porcine circovirus-associated disease (PCVAD) (1, 9, 10, 25). PCV1 and PCV2 have similar genomic organizations, with two major ambisense open reading frames (ORFs) (16). ORF1 (rep) encodes two viral replication-associated proteins, Rep and Rep′, by differential splicing (4, 6, 21, 22). The Rep and Rep′ proteins bind to specific sequences within the origin of replication (Ori) located in the intergenic region, and both are responsible for viral replication (5, 7, 8, 21, 23, 28, 29). ORF2 (cap) encodes the immunogenic capsid protein (Cap) (26). PCV1 and PCV2 share approximately 80%, 82%, and 62% nucleotide sequence identity in the Ori, rep, and cap, respectively (19).

In vitro studies using a reporter gene-based assay system showed that the replication factors of PCV1 and PCV2 are functionally interchangeable (2-6, 22), although this finding has not yet been validated in a live infectious-virus system. We have previously shown that chimeras of PCV in which cap has been exchanged between PCV1 and PCV2 are infectious both in vitro and in vivo (15), and an inactivated vaccine based on the PCV1-PCV2 cap (PCV1-cap2) chimera is used in the vaccination program against PCVAD (13, 15, 18, 27).

PCV1 replicates more efficiently than PCV2 in PK-15 cells (14, 15); thus, we hypothesized that the Ori or rep is directly responsible for the differences in replication efficiencies. The objectives of this study were to demonstrate that the Ori and rep are interchangeable between PCV1 and PCV2 in a live-virus system and to determine the effects of swapped heterologous replication factors on virus replication efficiency in vitro.

Construction of chimeric PCV infectious DNA clones.

PCV1 and PCV2a infectious DNA clones (11, 12, 15) were used as the genomic backbone for the construction of 4 new chimeric infectious-virus clones, PCV1-PCV2 Ori (PCV1-Ori2), PCV1-PCV2 rep (PCV1-rep2), PCV2-PCV1 Ori (PCV2-Ori1), and PCV2-PCV1 rep (PCV2-rep1), by overlap extension PCR (Fig. (Fig.1A).1A). The full-length chimeric genomes were assembled from two overlapping PCR fragments for chimera PCV1-Ori2 (Table (Table1,1, primers 3 plus 8 and 4 plus 7, PCV1 backbone) and chimera PCV2-Ori1 (primers 1 plus 6 and 2 plus 5, PCV2 backbone). Three fragments were used to assemble chimera PCV1-rep2 (primers 1 plus 15 and 2 plus 14, PCV2 backbone; primer 13 plus 16, PCV1 backbone) and chimera PCV2-rep1 (primers 3 plus 11 and 4 plus 10, PCV1 backbone; primer 9 plus 12, PCV2 backbone). Fusion PCR was used to assemble fragments of the chimeric DNA clones (primers 1 plus 2 or 3 plus 4). The chimeric fusion products were digested with KpnI or SacII and cloned into pCR2.1 or pBluescript II SK(+), respectively. Each chimeric clone was completely sequenced to confirm that no mutations had been introduced. Infectious-virus stocks of PCV1, PCV2, and the 4 chimeric viruses were generated by transfection of PK-15 cells with dimerized or concatamerized infectious clones (12, 15). PCV1 and PCV2 and all 4 chimeric infectious clones were found to produce infectious virus after transfection into PK-15 cells, and each infectious-virus stock was titrated by an immunofluorescence assay (12).

FIG. 1.
Schematic diagram of wild-type and chimeric PCVs and viral genomic DNA titer changes during in vitro replication. (A) Organization of wild-type and chimeric PCV infectious DNA clones. Genes encoding the viral capsid (cap) and replication-associated proteins ...
TABLE 1.
Oligonucleotide primers used in the construction of chimeric PCV infectious DNA clones

For growth characterization, PK-15 cells seeded in 24-well plates at 50% confluence were infected with a 0.5 multiplicity of infection of each virus, incubated for 1 h at 37°C, and washed three times with sterile Hanks' buffered salt solution. One ml of minimal essential medium (MEM) was then added to each well. Triplicate wells of infected cells for each virus were harvested every 12 h through 96 h postinfection (hpi), frozen and thawed three times, clarified at 2,500 × g at 4°C for 10 min, and stored at −80°C until titrated. At 12, 24, 36, 48, 60, 72, 84, or 96 hpi, the infectivity titers of each virus were determined (12) and compared by exact Kruskal-Wallis one-way analysis of variance (ANOVA), followed by Dunn's procedure for multiple comparisons.

The genomic copy numbers of viral DNA at 12 and 96 hpi were quantified. Viral genomes containing the rep gene of PCV2 were quantified with a published quantitative PCR (Q-PCR) (24). A 25-μl Q-PCR mixture (200 nM each primer [PCV2-83F, 5′-AAAAGCAAATGGGCTGCTAA-3′; PCV2-83R, 5′-TGGTAACCATCCCACCACTT-3′]; 200 μM deoxynucleoside triphosphate [dNTP], 5 mM MgCl2, and 1 μl DNA extract) was run in duplicate in a My iQ thermocycler. Viral genomes with the rep gene of PCV1 were quantified in duplicate in a 25-μl Q-PCR mixture (200 nM each primer [PCV1-qRepF, 5′-TGGAGAAGAAGTTGTTGT-3′; PCV1-qRepR, 5′-TCTACAGTCAATGGATACC-3′], 200 μM dNTP, 3 mM MgCl2, and 1 μl DNA extract), using a similar program. Net genomic titer changes in log10 genome copy numbers from 12 hpi to 96 hpi were compared among different viruses (Fig. (Fig.1B1B).

PCV1 replicates more efficiently than PCV2 in vitro.

The infectivity titer of PCV1 was found to be significantly greater (P ≤ 0.005) than that of PCV2 from 36 hpi to 96 hpi (Fig. (Fig.2A).2A). This is consistent with our previous studies showing that PCV1 replicates to higher titers in PK-15 cells than PCV2 (14, 15). The enhanced replication ability of PCV1 in PK-15 cells is likely due to the fact that PCV1 is already adapted to grow in PK-15 cells, since it was first isolated as a persistent contaminant of the PK-15 cell line (33).

FIG. 2.
One-step growth curve of wild-type and chimeric PCVs in PK-15 cells. PK-15 cells were infected with PCV1 (•), PCV2 (○), chimera PCV1-Ori2 ([filled square]), chimera PCV2-Ori1 (□), chimera PCV1-rep2 ([filled triangle]), or chimera PCV2-rep1 ([open triangle]) ...

Chimeric PCV2 viruses containing replication factors of PCV1 have enhanced replication in vitro.

Chimeric PCV2 viruses containing rep and the Ori of PCV1 were found to have increased replication efficiencies (Fig. (Fig.2B).2B). The infectious titers of chimera PCV2-rep1 virus were significantly higher (P ≤ 0.047) at 72 and 96 hpi than those of PCV2 (Fig. (Fig.2B).2B). By 96 hpi, cells infected with chimera PCV2-rep1 produced at least 10-fold-more infectious virus than cells infected with PCV2 (Fig. (Fig.2B).2B). The chimera PCV2-Ori1 infectivity titers were also increased compared to those of PCV2 prior to 72 hpi (Fig. (Fig.2B).2B). Conversely, chimeric PCV1 viruses containing the Ori or rep of PCV2 showed a general decrease in infectivity titers from 36 to 96 hpi compared to those for PCV1 (Fig. (Fig.2C),2C), although this was not as large as the increase observed for the PCV2 chimeric viruses.

Differences in the rep gene and Ori of PCV1 and PCV2 affect virus replication efficiencies.

In general, chimeric viruses based on the PCV1 backbone replicated to higher infectivity titers at an earlier time, and the titers remained higher throughout the study. Chimera PCV1-Ori2 achieved significantly higher infectivity titers than PCV2 at 36, 48, 72, and 84 hpi (P ≤ 0.035). The virus titers produced by the chimera PCV1-rep2 were significantly higher than those of PCV2 at 48, 60, 84, and 96 hpi (P ≤ 0.042) but were not significantly different from those of PCV1 (Fig. (Fig.2C).2C). The significant difference in net changes of viral genomic copy numbers (P = 0.027) (Fig. (Fig.1B)1B) from 12 to 96 hpi between chimeras PCV1-Ori2 and PCV1-rep2 is consistent with our finding that the production of infectious virus by chimeric viruses containing the PCV1 rep gene was significantly enhanced.

Possible explanations for the differences in replication efficiencies observed in this study include changes in gene expression and genome replication and altered interactions between cellular and viral proteins. The promoters for cap and rep, located within rep and the Ori, respectively (3), were exchanged in the chimeric viruses in this study. Rep is known to repress the rep promoter; thus, differences in binding between PCV1 Rep and PCV2 Rep could affect the levels of expression from heterologous promoters (20). Differences in the abilities of chimeric viruses to replicate viral genomic DNA could alter the amount and timing of infectious-virus production. However, the observed differences in infectivity titers are unlikely due to viral-DNA replication efficiencies alone, since the wild type and most chimeric viruses produced similar amounts of viral DNA genomes (Fig. (Fig.1B1B and data not shown).

PCV Cap is known to interact with Rep and several cellular proteins and thus may influence intracellular transport, encapsidation, and/or transcription of viral genes (17, 30). In our study, chimeric viruses with Cap and Rep of PCV2 were less efficient in the production of infectious virus. Compared to that produced by PCV1, significantly less infectious virus was produced at 36 and 84 hpi (P ≤ 0.035) by chimera PCV2-rep1 and at 36, 48, and 96 hpi (P ≤ 0.039) by chimera PCV2-Ori1 (Fig. (Fig.2B).2B). Furthermore, chimeric PCV1 viruses with either rep2 or Ori2 produced significantly more infectious virus (P ≤ 0.043) at most time points after 24 hpi than PCV2, but infectious-virus production was not significantly different from that of PCV1 (Fig. (Fig.2C).2C). Interestingly, the replication efficiency of the PCV1-cap2 chimera from our previous studies was decreased in comparison to those of PCV2 and chimera PCV2-cap1 (15), and two amino acid changes in Cap were shown to increase the replication efficiency of PCV2 in vitro (14). Thus, complex interactions between Cap, Rep, and cellular factors with viral DNA elements, including the Ori, are likely responsible for the observed differences in production of infectious virus.

In summary, by using a live chimeric-virus system, we provided direct proof of the results of earlier studies with reporter gene-based assays that the replication factors are completely exchangeable between PCV1 and PCV2. Most importantly, we demonstrated that both the rep gene and the Ori of PCV1 enhance replication of the PCV2-based chimeric viruses. It remains to be seen, however, whether or not enhanced replication ability in vitro has any effect on the in vivo pathogenicity of these chimeric viruses. Nevertheless, the results have important implications for engineering a chimeric PCV2 virus with enhanced replication capability for future vaccine development.

Acknowledgments

The PCV1 monoclonal antibody was a gift from Gordon Allan. We thank Stephen Werre for his assistance with statistical analysis, Barbara Dryman and Sara Smith for their technical assistance, and Yaowei Huang for his review of the manuscript.

This work was supported in part by a research grant from Fort Dodge Animal Health, Inc.

Footnotes

[down-pointing small open triangle]Published ahead of print on 23 June 2010.

REFERENCES

1. Allan, G. M., F. McNeilly, S. Kennedy, B. Daft, E. G. Clarke, J. A. Ellis, D. M. Haines, B. M. Meehan, and B. M. Adair. 1998. Isolation of porcine circovirus-like viruses from pigs with a wasting disease in the U. S. A. and Europe. J. Vet. Diagn. Invest. 10:3-10. [PubMed]
2. Bratanich, A. C., and A. Blanchetot. 2002. PCV2 replicase transcripts in infected porcine kidney (PK15) cells. Virus Genes 25:323-328. [PubMed]
3. Cheung, A. K. 2003. Comparative analysis of the transcriptional patterns of pathogenic and nonpathogenic porcine circoviruses. Virology 310:41-49. [PubMed]
4. Cheung, A. K. 2003. The essential and nonessential transcription units for viral protein synthesis and DNA replication of porcine circovirus type 2. Virology 313:452-459. [PubMed]
5. Cheung, A. K. 2004. Identification of an octanucleotide motif sequence essential for viral protein, DNA, and progeny virus biosynthesis at the origin of DNA replication of porcine circovirus type 2. Virology 324:28-36. [PubMed]
6. Cheung, A. K. 2004. Identification of the essential and non-essential transcription units for protein synthesis, DNA replication and infectious virus production of porcine circovirus type 1. Arch. Virol. 149:975-988. [PubMed]
7. Cheung, A. K. 2006. Rolling-circle replication of an animal circovirus genome in a theta-replicating bacterial plasmid in Escherichia coli. J. Virol. 80:8686-8694. [PMC free article] [PubMed]
8. Cheung, A. K. 2007. A stem-loop structure, sequence non-specific, at the origin of DNA replication of porcine circovirus is essential for termination but not for initiation of rolling-circle DNA replication. Virology 363:229-235. [PubMed]
9. Clark, E. G. 1997. Post-weaning multisystemic wasting syndrome, p. 499-501. In Proceedings of the American Association of Swine Practitioners, 28th Annual Meeting. American Association of Swine Practitioners, Perry, IA.
10. Ellis, J., L. Hassard, E. Clark, J. Harding, G. Allan, P. Willson, J. Strokappe, K. Martin, F. McNeilly, B. Meehan, D. Todd, and D. Haines. 1998. Isolation of circovirus from lesions of pigs with postweaning multisystemic wasting syndrome. Can. Vet. J. 39:44-51. [PMC free article] [PubMed]
11. Fenaux, M., P. G. Halbur, M. Gill, T. E. Toth, and X. J. Meng. 2000. Genetic characterization of type 2 porcine circovirus (PCV-2) from pigs with postweaning multisystemic wasting syndrome in different geographic regions of North America and development of a differential PCR-restriction fragment length polymorphism assay to detect and differentiate between infections with PCV-1 and PCV-2. J. Clin. Microbiol. 38:2494-2503. [PMC free article] [PubMed]
12. Fenaux, M., P. G. Halbur, G. Haqshenas, R. Royer, P. Thomas, P. Nawagitgul, M. Gill, T. E. Toth, and X. J. Meng. 2002. Cloned genomic DNA of type 2 porcine circovirus is infectious when injected directly into the liver and lymph nodes of pigs: characterization of clinical disease, virus distribution, and pathologic lesions. J. Virol. 76:541-551. [PMC free article] [PubMed]
13. Fenaux, M., T. Opriessnig, P. G. Halbur, F. Elvinger, and X. J. Meng. 2004. A chimeric porcine circovirus (PCV) with the immunogenic capsid gene of the pathogenic PCV type 2 (PCV2) cloned into the genomic backbone of the nonpathogenic PCV1 induces protective immunity against PCV2 infection in pigs. J. Virol. 78:6297-6303. [PMC free article] [PubMed]
14. Fenaux, M., T. Opriessnig, P. G. Halbur, F. Elvinger, and X. J. Meng. 2004. Two amino acid mutations in the capsid protein of type 2 porcine circovirus (PCV2) enhanced PCV2 replication in vitro and attenuated the virus in vivo. J. Virol. 78:13440-13446. [PMC free article] [PubMed]
15. Fenaux, M., T. Opriessnig, P. G. Halbur, and X. J. Meng. 2003. Immunogenicity and pathogenicity of chimeric infectious DNA clones of pathogenic porcine circovirus type 2 (PCV2) and nonpathogenic PCV1 in weanling pigs. J. Virol. 77:11232-11243. [PMC free article] [PubMed]
16. Finsterbusch, T., and A. Mankertz. 2009. Porcine circoviruses—small but powerful. Virus Res. 143:177-183. [PubMed]
17. Finsterbusch, T., T. Steinfeldt, K. Doberstein, C. Rodner, and A. Mankertz. 2009. Interaction of the replication proteins and the capsid protein of porcine circovirus type 1 and 2 with host proteins. Virology 386:122-131. [PubMed]
18. Gillespie, J., N. M. Juhan, J. DiCristina, K. F. Key, S. Ramamoorthy, and X. J. Meng. 2008. A genetically engineered chimeric vaccine against porcine circovirus type 2 (PCV2) is genetically stable in vitro and in vivo. Vaccine 26:4231-4236. [PubMed]
19. Mankertz, A., R. Caliskan, K. Hattermann, B. Hillenbrand, P. Kurzendoerfer, B. Mueller, C. Schmitt, T. Steinfeldt, and T. Finsterbusch. 2004. Molecular biology of porcine circovirus: analyses of gene expression and viral replication. Vet. Microbiol. 98:81-88. [PubMed]
20. Mankertz, A., and B. Hillenbrand. 2002. Analysis of transcription of porcine circovirus type 1. J. Gen. Virol. 83:2743-2751. [PubMed]
21. Mankertz, A., and B. Hillenbrand. 2001. Replication of porcine circovirus type 1 requires two proteins encoded by the viral rep gene. Virology 279:429-438. [PubMed]
22. Mankertz, A., B. Mueller, T. Steinfeldt, C. Schmitt, and T. Finsterbusch. 2003. New reporter gene-based replication assay reveals exchangeability of replication factors of porcine circovirus types 1 and 2. J. Virol. 77:9885-9893. [PMC free article] [PubMed]
23. Mankertz, A., F. Persson, J. Mankertz, G. Blaess, and H. J. Buhk. 1997. Mapping and characterization of the origin of DNA replication of porcine circovirus. J. Virol. 71:2562-2566. [PMC free article] [PubMed]
24. McIntosh, K. A., A. Tumber, J. C. Harding, S. Krakowka, J. A. Ellis, and J. E. Hill. 2009. Development and validation of a SYBR green real-time PCR for the quantification of porcine circovirus type 2 in serum, buffy coat, feces, and multiple tissues. Vet. Microbiol. 133:23-33. [PubMed]
25. Meehan, B. M., F. McNeilly, D. Todd, S. Kennedy, V. A. Jewhurst, J. A. Ellis, L. E. Hassard, E. G. Clark, D. M. Haines, and G. M. Allan. 1998. Characterization of novel circovirus DNAs associated with wasting syndromes in pigs. J. Gen. Virol. 79:2171-2179. [PubMed]
26. Nawagitgul, P., I. Morozov, S. R. Bolin, P. A. Harms, S. D. Sorden, and P. S. Paul. 2000. Open reading frame 2 of porcine circovirus type 2 encodes a major capsid protein. J. Gen. Virol. 81:2281-2287. [PubMed]
27. Segales, J., A. Urniza, A. Alegre, T. Bru, E. Crisci, M. Nofrarias, S. Lopez-Soria, M. Balasch, M. Sibila, Z. Xu, H. J. Chu, L. Fraile, and J. Plana-Duran. 2009. A genetically engineered chimeric vaccine against porcine circovirus type 2 (PCV2) improves clinical, pathological and virological outcomes in postweaning multisystemic wasting syndrome affected farms. Vaccine 27:7313-7321. [PubMed]
28. Steinfeldt, T., T. Finsterbusch, and A. Mankertz. 2006. Demonstration of nicking/joining activity at the origin of DNA replication associated with the rep and rep′ proteins of porcine circovirus type 1. J. Virol. 80:6225-6234. [PMC free article] [PubMed]
29. Steinfeldt, T., T. Finsterbusch, and A. Mankertz. 2001. Rep and Rep′ protein of porcine circovirus type 1 bind to the origin of replication in vitro. Virology 291:152-160. [PubMed]
30. Timmusk, S., C. Fossum, and M. Berg. 2006. Porcine circovirus type 2 replicase binds the capsid protein and an intermediate filament-like protein. J. Gen. Virol. 87:3215-3223. [PubMed]
31. Tischer, I., H. Gelderblom, W. Vettermann, and M. A. Koch. 1982. A very small porcine virus with circular single-stranded DNA. Nature 295:64-66. [PubMed]
32. Tischer, I., W. Mields, D. Wolff, M. Vagt, and W. Griem. 1986. Studies on epidemiology and pathogenicity of porcine circovirus. Arch. Virol. 91:271-276. [PubMed]
33. Tischer, I., R. Rasch, and G. Tochtermann. 1974. Characterization of papovavirus- and picornavirus-like particles in permanent pig kidney cell lines. Zentralbl. Bakteriol. Orig. A 226:153-167. [PubMed]
34. Todd, D., M. Bendinelli, P. Biagini, S. Hino, S. Mamkertz, S. Mishiro, C. Niel, H. Okamoto, S. Raidal, B. W. Ritchie, and G. C. Teo. 2005. Circoviridae, p. 327-334. In C. M. Fauquet, M. A. Mayo, J. Maniloff, U. Desselberger, and L. A. Ball (ed.), Virus taxonomy: eighth report of the international committee on taxonomy of viruses. Elsevier Academic Press, San Diego, CA.

Articles from Journal of Virology are provided here courtesy of American Society for Microbiology (ASM)