1 Department of Molecular Biosciences, Section of Veterinary Immunology and Virology, Swedish University of Agricultural Sciences, Biomedical Centre, PO Box 588, SE-751 23 Uppsala, Sweden
2 Veterinary Sciences Division, Virology Section, Department of Agriculture and Rural Development for Northern Ireland, Stormont, Belfast BT4 3SD, UK
3 Department of Veterinary Science, Queen's University Belfast, Stormont, Belfast BT4 3SD, UK
Correspondence
Frida Hasslung
frida.hasslung{at}vmm.slu.se
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The small (1759 nt), circular genome of PCV-2 is analogous to plasmid DNA, which is known to exhibit immunostimulatory activity in vertebrates (Tighe et al., 1998). This immunostimulatory activity includes induction of cytokine production, proliferation and immunoglobulin secretion by B-cells as well as enhanced NK-cell activity and is ascribed to the relatively high content of unmethylated CpG dinucleotides in plasmids and other forms of bacterial DNA (Krieg, 2002
; Sato et al., 1996
; Yamamoto et al., 1992
). An unmethylated CpG dinucleotide flanked by two 5' purines and two 3' pyrimidines was referred to initially as a stimulatory CpG motif (Sato et al., 1996
). Using synthetic oligodeoxynucleotides (ODNs), it has been demonstrated that alteration of the flanking nucleotides affects the immune stimulatory capacity. However, optimal flanking bases vary between species and with the immune parameter studied (Klinman et al., 2002
; Krieg, 2002
; Mutwiri et al., 2003
). Furthermore, dissection of the requirements for immune stimulatory activity (CpG-S) has revealed DNA motifs that neutralize (CpG-N) or inhibit immune stimulation. The sequence of inhibitory motifs can resemble those of stimulatory motifs but the most efficient inhibitors contain a G tetramer or repeated clusters of CG sequences (Krieg et al., 1998
; Pisetsky & Reich, 2000
; Stunz et al., 2002
; Zhao et al., 2000
). In addition, the position of the inhibitory sequence in relation to a stimulatory sequence on the same strand of DNA is considered of importance with respect to the net effect of the ODN (Yamada et al., 2002
).
Studies in mice and man suggest that CpG DNA interacts with Toll-like receptor 9, which, in the human system, is expressed by B-cells and a subpopulation of dendritic cells (Krieg, 2002). PCV-2 has been demonstrated to accumulate in the cytoplasm of monocytes/macrophages and dendritic cells of infected pigs in the absence of any evidence of active virus replication (Allan et al., 1998
; Ellis et al., 1998
). Since these cell types are efficient cytokine producers and play an important role in directing the host immune response, they are important targets for virus evasion.
In this study, the genome of PCV-2 was examined for CpG content and five sequences, each 20 nt long with central CpG motifs, selected for their similarity to known stimulatory and inhibitory ODNs, were analysed for their ability to modulate the production of the antiviral cytokine IFN- by porcine leukocytes.
![]() |
METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
Lipofection.
Salmon sperm DNA, ODNs, plasmid DNA and viruses were, where indicated, pretreated with lipofectin (Life Technologies). For lipofectin treatment, lipofectin (10 µg ml-1) was incubated for 1 h at room temperature in RPMI 1640 medium (BioWhittaker) before mixing with an equal volume of medium containing salmon sperm DNA, ODN, plasmid or virus. Samples were pretreated for 15 min and were then added to cell cultures, as described below. When cells were cocultured with two types of inducers, these were incubated with lipofectin either individually or together before addition to cell cultures. In all cases, the final concentration of lipofectin was 2·5 µg ml-1 in medium.
Cell cultures and induction of IFN-.
Blood samples were collected from conventionally reared Yorkshire pigs or Yorkshire crosses, aged 812 weeks and housed at the University Research Station Funbo, Lövsta, Uppsala, Sweden. Blood was collected from the vena cava cranialis in evacuated test tubes (B.-D. Vacutainer) with heparin (143 USP units) and PBMCs were purified from blood by FicollPaque (Amersham Pharmacia) density gradient centrifugation (30 min at 500 g). PBMCs were suspended in growth medium (RPMI 1640 medium with 20 mM HEPES buffer, supplemented with 2 mM L-glutamine, 200 IU penicillin ml-1, 100 µg streptomycin ml-1 and 5x10-5 M 2-mercaptoethanol) and heat-inactivated FCS (Life Technologies) at a final concentration of 5 % (v/v) in cell culture. Cultures for cytokine induction were established in flat-bottomed, 96-well plates (Nunc). Each type of inducer was tested in triplicate cultures, consisting of 0·1 ml PBMCs (5x106 ml-1) and 0·1 ml inducer at the following final concentrations: 2·5 µg pcDNA3 ml-1, ADV corresponding to 103 ID50 ml-1 before UV-inactivation, live SV preparation diluted 1 : 10 and ODNs at 1, 5, 25 or 75 µg ml-1. After 20 h of incubation at 37 °C and 7 % CO2, supernatants from the replicates were pooled and stored at -20 °C until further analysis.
Detection of IFN-.
IFN- in cell culture supernatants was quantified by a dissociation-enhanced lanthanide fluoro-immunoasssay (DELFIA), as described previously (Artursson et al., 1995
). DELFIA, which is based on two mAbs directed to porcine IFN-
, had a detection limit of 0·3 U IFN-
ml-1. The concentration of IFN-
was determined by comparison with a laboratory standard of natural porcine IFN-
and the results are given as U IFN-
ml-1. Results from experiments in which ODNs were cocultivated are given as percentage, calculated from the formula: 100x(U IFN-
ml-1 in cultures with both inducers)/(U IFN-
ml-1 in cultures with a single inducer). All values are the mean±SEM for four animals.
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Regardless of the concentration used, pretreatment of ODNs with lipofectin was necessary to achieve IFN- production. ODNs PCV-2/4 and PCV-2/5 were the strongest inducers but ODNs PCV-2/2 and PCV-2/3 also induced IFN-
production (Fig. 1
). IFN-
induction increased with concentration of the respective ODN and 25 µg ODN ml-1 consistently induced the highest amounts of IFN-
. At this concentration, ODNs PCV-2/4 and PCV-2/5 induced similar levels of IFN-
as lipofected plasmid DNA (pcDNA3: pig 1, 316 U IFN-
ml-1; pig 2, 653 U IFN-
ml-1; pig 3, 386 U IFN-
ml-1; pig 4, 368 U IFN-
ml-1). Although the overall IFN-
-producing capacity varied between individual pigs, their responses to the various inducers showed the same internal relationship. However, of particular significance was that one of the ODNs, PCV-2/1, did not induce IFN-
production by PBMCs from any of the pigs tested (<0·3 U IFN-
ml-1), regardless of pretreatment or not with lipofectin. Consequently, the possible inhibitory activity of ODN PCV-2/1 was studied further.
|
To study whether the inhibitory effect of ODN PCV-2/1 observed was due to competition between nucleic acid preparations for incorporation into liposomes, the effects of ODN PCV-2/1 were compared to that of salmon sperm DNA. Liposomes consisting of ODN PCV-2/5 (25 µg ml-1) or salmon sperm DNA (50 µg ml-1) alone induced 251±34·4 and 12·2±4·8 U IFN- ml-1, respectively. Liposomes made up of both PCV-2/5 (25 µg ml-1) and salmon sperm DNA (25 µg ml-1) induced 74·4±10·4 U IFN-
ml-1, whereas no IFN-
(<0·3 U ml-1) was induced by liposomes consisting of ODN PCV-2/5 (25 µg ml-1) in combination with ODN PCV-2/1 (25 µg ml-1). Thus, the decrease in IFN-
production can be explained only in part by a reduced concentration of ODN PCV-2/5 in the liposomes and a specific inhibitory effect of ODN PCV-2/1 is indicated.
In the following experiment, PCV-2/5 and PCV-2/1 were pretreated with lipofectin separately before addition to the cultures. PCV-2/5 induced 363±192 U IFN- ml-1 in the absence of PCV-2/1. When mixed with an equal concentration of PCV-2/1 (25 µg ml-1), the induction of IFN-
by PCV-2/5 was decreased to 13±6 U IFN-
ml-1 and induction was abolished totally (<0·3 U IFN-
ml-1 culture supernatant) when PCV-2/5 was tested in combination with PCV-2/1 at a threefold higher concentration (75 µg ml-1). Thus, no obvious difference between individual or combined incubation with lipofectin was observed and, in subsequent experiments, inducers needing pretreatment with lipofectin were incubated separately.
PCV-2/1 partially inhibits IFN- induction by some nonrelated ODNs
To study further the inhibitory capacity of PCV-2/1, three additional ODNs with the ability to induce IFN- production in the absence of lipofectin (Domeika, 2003
) were synthesized. These ODNs consist of a central CpG motif and G repeats in their 5' and 3' ends (phosphodiester ODN D25 or the phophodiester/phosphorothioate chimeras ODN D19 and 2216, Table 1
). According to previous results, ODNs D19 and D25 were used at the concentration 25 µg ml-1, and ODN 2216 at 5 µg ml-1, concentrations that are optimal for IFN-
induction in porcine PBMCs (Domeika, 2003
). The IFN-
induction of these ODNs following cocultivation with PCV-2/1 (25 and 75 µg ml-1) in the absence of lipofectin is shown as percentage of these values (Fig. 2
).
|
PCV-2/1 inhibits ODNs that require pretreatment with lipofectin
ODN H and its antisense counterpart ODN I (Table 1) have been used previously as efficient IFN-
inducers in both ss and ds forms, provided they are pretreated with lipofectin (Magnusson et al., 2001b
). Modification of ODN H by addition of a poly(G) sequence at the 3' end increases its IFN-
-inducing capacity but does not circumvent the need for lipofectin (Domeika, 2003
). To study the influence of poly(G) sequences on the inhibitory effect of PCV-2/1, these lipofected ODNs were used to induce IFN-
in porcine PBMCs alone or in combination with lipofected PCV-2/1 (Fig. 2
).
In the absence of PCV-2/1, ODN H induced a mean value of 398±150 U IFN- ml-1; ODN I induced 313±126 U IFN-
ml-1 and the ds form HI induced 716±248 U IFN-
ml-1. When any of these three ODNs were pretreated separately with lipofectin and mixed with PCV-2/1 at equal concentrations (25 µg ml-1), induction of IFN-
was abolished totally. ODN HPoly-G (25 µg ml-1) induced a mean value of 1124±254 U IFN-
ml-1 in the absence of PCV-2/1. After pretreatment with lipofectin, it was mixed with PCV-2/1 at two different concentrations (25 and 75 µg ml-1). At the lower concentration, IFN-
production was reduced and at the higher concentration it was abolished totally. Thus, PCV-2/1 seemed to efficiently inhibit ss as well as ds ODNs that required pretreatment with lipofectin to induce IFN-
. PCV-2/1 could also inhibit ODN H after modification by addition of a poly(G) sequence to its 3' end.
PCV-2/1 inhibits IFN- induction by ADV and pcDNA3 but not SV
Viruses are well known inducers of IFN- and the effect of PCV-2/1 was, therefore, studied using two different virus preparations: inactivated ADV and live SV. For comparison, lipofected pcDNA3 was used as a DNA IFN-
-inducing positive control. As in the previous experiments, PCV-2/1 was tested at two different concentrations (Fig. 3
). In the absence of PCV-2/1, SV induced mean values of 109±28 U IFN-
ml-1, ADV induced 390±149 U IFN-
ml-1 and pcDNA3 induced 952±245 U IFN-
ml-1. When mixed with live SV, no consistent effect of PCV-2/1 on the levels of IFN-
produced was observed. In contrast, however, IFN-
production induced by ADV was inhibited clearly by PCV-2/1 at both concentrations. PCV-2/1 in combination with pcDNA3 resulted in a partial inhibition of IFN-
production induced by the plasmid, with the higher concentration of PCV-2/1 being most effective. Since pcDNA3 does not induce IFN-
production in the absence of lipofectin, only lipofected samples were tested. Pretreatment of the virus inducers with lipofectin did not influence the inhibitory effect of PCV-2/1 (data not shown).
|
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Four of the ODNs induced IFN- production at levels similar to that induced by plasmid DNA. Thus, one of the presumed inhibitory ODNs (PCV-2/2) did induce IFN-
production despite the presence of two inhibitory motifs, CGG, spaced by 3 nt. This unexpected capacity could be due to the positioning of the motifs in relation to each other, to the flanking nucleotides and the lengths of the entire ODN. Alternatively, this CGG motif might simply not be an inhibitory motif for IFN-
induction in pigs. The fifth ODN (PCV-2/1), which was selected due to its content of CG repeats did not induce detectable levels of IFN-
and, of particular significance, was demonstrated to inhibit IFN-
production induced by the other ODNs representing parts of the PCV-2 genome. The inhibitory activity of PCV-2/1 was demonstrated further against two unrelated ODNs (H and its complementary strand I), which lost their IFN-
-inducing capacity in the presence of PCV-2/1. Thus, PCV-2/1 inhibited almost completely the IFN-
induction by phosphodiester ODNs, in both their ss and their ds forms.
In a previous study on IFN- induction by plasmid DNA, lipofected salmon sperm DNA was used as a neutral control DNA preparation that was shown not to induce IFN-
production in porcine cells (Magnusson et al., 2001a
). Liposomes formed with equal amounts of salmon sperm DNA and ODN PCV-2/5 were less efficient inducers of IFN-
than liposomes containing only ODN PCV-2/5, but the presence of salmon sperm DNA in the liposomes did not inhibit IFN-
-inducing capacity, as demonstrated in the presence of ODN PCV-2/1. Thus, the inhibitory effect of ODN PCV-2/1 could not be explained by competition between inert and stimulatory DNA in the liposomes. It is notable that the inhibitory motif identified was also inhibitory as a ds form, representing the replicative form of the viral DNA. Indeed, the complementary sequence (PCV-2/1 C) showed a similar, or even higher, inhibitory capacity. However, further studies are needed to compare and evaluate the inhibitory effects of ds and ss forms of the ODNs.
Most CpG phosphodiester ODNs need preincubation with lipofectin to induce IFN- in cultures of human and porcine PBMCs (Magnusson et al., 2001b
). In addition, to protect against nucleases, lipofectin also facilitates the cellular uptake of incorporated molecules (Xu & Szoka, 1996
). Exchange of phosphodiester to phosphorothioate nucleotides increases resistance to nucleases (Stein et al., 1988
) and two ODNs with a chimeric backbone (ODN D19 and ODN 2216) both induced IFN-
efficiently without preincubation with lipofectin. Of particular significance, ODN PCV-2/1 was unable to inhibit IFN-
production induced by ODN D19 but reduced ODN 2216-induced levels of IFN-
by 50 % or more, both in the presence and in the absence of lipofectin. The discrepancy in inhibitory effect on ODN 2216 and D19 could be explained by the fact that the two ODNs have different concentration optima for IFN-
induction (Domeika, 2003
) and ODN 2216 was used at a fivefold lower concentration (5 µg ml-1) than ODN D19 (25 µg ml-1). Nevertheless, these results indicate that PCV-2/1 activity is not dependent on lipofectin to exert its inhibitory effect. IFN-
induction by ODN D25, which contains the same base sequence as ODN D19 but is constructed with a phosphodiester backbone, was only inhibited partially by PCV-2/1. This ODN is known to activate the genes for IL-6, IL-12 and TNF-
in porcine PBMCs (Kamstrup et al., 2001
) and contains a poly(G) sequence, which, in other species, has been demonstrated to mediate uptake via scavenger receptors (Dalpke et al., 2002
; Peiser et al., 2002
). However, the addition of a poly(G) sequence to ODN H did not substitute the need for pretreatment with lipofectin to induce IFN-
production by this ODN and reduced only marginally the inhibitory effect of PCV-2/1. Thus, no single ODN characteristic could explain to what level PCV-2/1 inhibited the IFN-
induction by other ODNs. General modifications of the ODNs, such as addition of a poly(G) sequence or conversion to a phosphorothioate backbone, which are known to increase the immune stimulatory activity of the ODN even in the absence of a CpG motif (Hartmann et al., 1996
; Krieg, 2002
), seemed, however, to counteract the inhibitory effect of PCV-2/1.
One possible explanation to the variable effect of PCV-2/1 could be that particular cell types are activated to produce IFN- by the different ODNs. In human PBMCs, two populations of IFN-
/
-producing cells are recognized: monocytes and cells of plasmacytoid dendritic cell (PDC) origin, the natural IFN-producing cells (NIPCs) (Colonna et al., 2002
). In the pig, cells with many characteristics similar to NIPCs have been demonstrated after induction with transmissible gastroenteritis virus (TGEV) or ADV (Artursson et al., 1992
; Nowacki et al., 1993
; Nowacki & Charley, 1993
). When human PBMCs are exposed to SV, monocytes in addition to NIPCs produce IFN-
, whereas only NIPCs respond to herpes simplex virus, pcDNA3 (Vallin et al., 1999
) or CpG-ODN (Magnusson et al., 2001b
). In particular, ODN 2216 is a potent inducer of IFN-
that activates PDC/NIPC cells selectively (Krug et al., 2001
). In support of this hypothesis, the induction of IFN-
by SV was not affected by PCV-2/1, while the induction by ADV was reduced substantially. In addition, the production of IFN-
induced by plasmid DNA (pcDNA3) was reduced clearly upon addition of PCV-2/1. Current phenotyping of porcine cells suggests that cells responding with IFN-
production at exposure to TGEV (A. Summerfield, Institute of Virology and Immunoprophylaxis, Mittelhäusern, Switzerland, personal communication) as well as to ODN 2216 and pcDNA3 (K. Domeika, M. Magnusson, M.-L. Eloranta, L. Fuxler, G. V. Alm and C. Fossum, unpublished results) constitute a subset of dendritic cells with great similarities to human NIPCs. In contrast, porcine monocyte-derived dendritic cells produce IFN-
following exposure to SV but are nonresponsive to ADV and plasmid DNA (Johansson et al., 2003
). Thus, the inhibitory activity of PCV-2/1 seems to be more pronounced for IFN-
production by PDCs than by monocytes.
In addition to its direct antiviral effect, IFN- contributes to the specific immune response to virus infections (Le Bon & Tough, 2002
). Therefore, PDCs comprise a potentially important strategic target for virus evasion. To date, the site of replication of PCV-2 is unknown but the virus accumulates in the cytoplasm of dendritic cells and macrophages in the absence of any evidence of active virus replication (Allan & Ellis, 2000
). In this study, the demonstration of IFN-
-inducing as well as inhibitory motifs in the genome of PCV-2 could, therefore, contribute to the understanding of the pathogenesis of PCV-2-associated syndromes, such as PMWS.
In general, viral DNA appears to have evolved in the direction towards either lower CpG content or a ratio between CpG-S and CpG-N that is biased to substantially more CpG-N motifs (Karlin et al., 1994; Krieg et al., 1998
; Sun et al., 1997
). Thus, the evolution of some viruses seems to aim at avoidance of immune activation via CpG. One such example is adenovirus type 2, which has substantially more CpG-N motifs than adenovirus type 12. This renders its genome inert or even inhibitory for cytokine induction and has been suggested to contribute to the establishment of a persistent adenovirus infection (Krieg et al., 1998
). Interestingly, another member of the family Circoviridae, chicken anaemia virus (CAV), causes severe immunosuppression in young chickens (Rosenberger & Cloud, 1998
). As in the case of PCV-2, the pathogenesis of CAV is not understood fully but the virus has been shown to interfere with the transcription of IFN-
and IFN-
mRNA (Ragland et al., 2002
). The genome of CAV has a high content of sequences with striking similarities to the inhibitory ODN PCV-2/1. These sequences may, therefore, play a role in the pathogenesis of CAV and could possibly explain the immunosuppressive effect of both of these viruses. The mechanisms of this action are not understood yet and further studies are needed to evaluate this hypothesis.
![]() |
ACKNOWLEDGEMENTS |
---|
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Allan, G. M., McNeilly, F., Cassidy, J. P., Reilly, G. A., Adair, B., Ellis, W. A. & McNulty, M. S. (1995). Pathogenesis of porcine circovirus; experimental infections of colostrum deprived piglets and examination of pig foetal material. Vet Microbiol 44, 4964.[CrossRef][Medline]
Allan, G. M., McNeilly, F., Kennedy, S., Daft, B., Clarke, E. G., Ellis, J. A., Haines, D. M., Meehan, B. M. & Adair, B. M. (1998). Isolation of porcine circovirus-like viruses from pigs with a wasting disease in the USA and Europe. J Vet Diagn Invest 10, 310.[Medline]
Allan, G. M., Kennedy, S., McNeilly, F., Foster, J. C., Ellis, J. A., Krakowka, S. J., Meehan, B. M. & Adair, B. M. (1999). Experimental reproduction of severe wasting disease by co-infection of pigs with porcine circovirus and porcine parvovirus. J Comp Pathol 121, 111.[CrossRef][Medline]
Allan, G. M., McNeilly, E., Kennedy, S., Meehan, B., Moffett, D., Malone, F., Ellis, J. & Krakowka, S. (2000). PCV-2-associated PDNS in Northern Ireland in 1990. Porcine dermatitis and nephropathy syndrome. Vet Rec 146, 711712.
Artursson, K., Gobl, A., Lindersson, M., Johansson, M. & Alm, G. (1992). Molecular cloning of a gene encoding porcine interferon-. J Interferon Res 12, 153160.[Medline]
Artursson, K., Lindersson, M., Varela, N., Scheynius, A. & Alm, G. V. (1995). Interferon- production and tissue localization of interferon-
/
producing cells after intradermal administration of Aujeszky's disease virus-infected cells in pigs. Scand J Immunol 41, 121129.[Medline]
Choi, J., Stevenson, G. W., Kiupel, M., Harrach, B., Anothayanontha, L., Kanitz, C. L. & Mittal, S. K. (2002). Sequence analysis of old and new strains of porcine circovirus associated with congenital tremors in pigs and their comparison with strains involved with postweaning multisystemic wasting syndrome. Can J Vet Res 66, 217224.[Medline]
Colonna, M., Krug, A. & Cella, M. (2002). Interferon-producing cells: on the front line in immune responses against pathogens. Curr Opin Immunol 14, 373379.[CrossRef][Medline]
Dalpke, A. H., Zimmermann, S., Albrecht, I. & Heeg, K. (2002). Phosphodiester CpG oligonucleotides as adjuvants: polyguanosine runs enhance cellular uptake and improve immunostimulative activity of phosphodiester CpG oligonucleotides in vitro and in vivo. Immunology 106, 102112.[CrossRef][Medline]
Domeika, K. (2003). Porcine immunoregulatory cytokines with special reference to their induction by CpG-containing DNA. In Department of Veterinary Microbiology, Section for Immunology. Uppsala: Swedish University of Agricultural Sciences.
Ellis, J., Hassard, L., Clark, E. & 9 other authors (1998). Isolation of circovirus from lesions of pigs with postweaning multisystemic wasting syndrome. Can Vet J 39, 4451.[Medline]
Harding, J. C., Clark, E. G., Strokappe, J. H., Willson, P. I. & Ellis, J. A. (1998). Postweaning multisystemic wasting syndrome: epidemiology and clinical presentation. Swine Health Prod 6, 249254.
Hartmann, G., Krug, A., Waller-Fontaine, K. & Endres, S. (1996). Oligodeoxynucleotides enhance lipopolysaccharide-stimulated synthesis of tumor necrosis factor: dependence on phosphorothioate modification and reversal by heparin. Mol Med 2, 429438.[Medline]
Jarrossay, D., Napolitani, G., Colonna, M., Sallusto, F. & Lanzavecchia, A. (2001). Specialization and complementarity in microbial molecule recognition by human myeloid and plasmacytoid dendritic cells. Eur J Immunol 31, 33883393.[CrossRef][Medline]
Johansson, E., Domeika, K., Berg, M., Alm, G. V. & Fossum, C. (2003). Characterisation of porcine monocyte-derived dendritic cells according to their cytokine profile. Vet Immunol Immunopathol 91, 183197.[CrossRef][Medline]
Kamstrup, S., Verthelyi, D. & Klinman, D. M. (2001). Response of porcine peripheral blood mononuclear cells to CpG-containing oligodeoxynucleotides. Vet Microbiol 78, 353362.[CrossRef][Medline]
Karlin, S., Ladunga, I. & Blaisdell, B. E. (1994). Heterogeneity of genomes: measures and values. Proc Natl Acad Sci U S A 91, 1283712841.
Kennedy, S., Moffett, D., McNeilly, F., Meehan, B., Ellis, J., Krakowka, S. & Allan, G. M. (2000). Reproduction of lesions of postweaning multisystemic wasting syndrome by infection of conventional pigs with porcine circovirus type 2 alone or in combination with porcine parvovirus. J Comp Pathol 122, 924.[CrossRef][Medline]
Klinman, D. M., Takeshita, F., Gursel, I., Leifer, C., Ishii, K. J., Verthelyi, D. & Gursel, M. (2002). CpG DNA: recognition by and activation of monocytes. Microbes Infect 4, 897901.[CrossRef][Medline]
Krakowka, S., Ellis, J. A., Meehan, B., Kennedy, S., McNeilly, F. & Allan, G. (2000). Viral wasting syndrome of swine: experimental reproduction of postweaning multisystemic wasting syndrome in gnotobiotic swine by coinfection with porcine circovirus 2 and porcine parvovirus. Vet Pathol 37, 254263.
Krakowka, S., Ellis, J. A., McNeilly, F., Ringler, S., Rings, D. M. & Allan, G. (2001). Activation of the immune system is the pivotal event in the production of wasting disease in pigs infected with porcine circovirus-2 (PCV-2). Vet Pathol 38, 3142.
Krieg, A. M. (2002). CpG motifs in bacterial DNA and their immune effects. Annu Rev Immunol 20, 709760.[CrossRef][Medline]
Krieg, A. M., Wu, T., Weeratna, R., Efler, S. M., Love-Homan, L., Yang, L., Yi, A. K., Short, D. & Davis, H. L. (1998). Sequence motifs in adenoviral DNA block immune activation by stimulatory CpG motifs. Proc Natl Acad Sci U S A 95, 1263112636.
Krug, A., Rothenfusser, S., Hornung, V., Jahrsdorfer, B., Blackwell, S., Ballas, Z. K., Endres, S., Krieg, A. M. & Hartmann, G. (2001). Identification of CpG oligonucleotide sequences with high induction of IFN-/
in plasmacytoid dendritic cells. Eur J Immunol 31, 21542163.[CrossRef][Medline]
Le Bon, A. & Tough, D. F. (2002). Links between innate and adaptive immunity via type I interferon. Curr Opin Immunol 14, 432436.[CrossRef][Medline]
Magnusson, M., Johansson, E., Berg, M., Eloranta, M. L., Fuxler, L. & Fossum, C. (2001a). The plasmid pcDNA3 differentially induces production of interferon- and interleukin-6 in cultures of porcine leukocytes. Vet Immunol Immunopathol 78, 4556.[CrossRef][Medline]
Magnusson, M., Magnusson, S., Vallin, H., Ronnblom, L. & Alm, G. V. (2001b). Importance of CpG dinucleotides in activation of natural IFN--producing cells by a lupus-related oligodeoxynucleotide. Scand J Immunol 54, 543550.[CrossRef][Medline]
Mankertz, A. & Hillenbrand, B. (2001). Replication of porcine circovirus type 1 requires two proteins encoded by the viral rep gene. Virology 279, 429438.[CrossRef][Medline]
Meehan, B. M., McNeilly, F., Todd, D. & 7 other authors (1998). Characterization of novel circovirus DNAs associated with wasting syndromes in pigs. J Gen Virol 79, 21712179.[Abstract]
Mutwiri, G., Pontarollo, R., Babiuk, S. & 12 other authors (2003). Biological activity of immunostimulatory CpG DNA motifs in domestic animals. Vet Immunol Immunopathol 91, 89103.[CrossRef][Medline]
Nowacki, W. & Charley, B. (1993). Enrichment of coronavirus-induced interferon-producing blood leukocytes increases the interferon yield per cell: a study with pig leukocytes. Res Immunol 144, 111120.[Medline]
Nowacki, W., Cederblad, B., Renard, C., La Bonnardiere, C. & Charley, B. (1993). Age-related increase of porcine natural interferon producing cell frequency and of interferon yield per cell. Vet Immunol Immunopathol 37, 113122.[CrossRef][Medline]
Peiser, L., Mukhopadhyay, S. & Gordon, S. (2002). Scavenger receptors in innate immunity. Curr Opin Immunol 14, 123128.[CrossRef][Medline]
Pisetsky, D. S. & Reich, C. F. (2000). Inhibition of murine macrophage IL-12 production by natural and synthetic DNA. Clin Immunol 96, 198204.[CrossRef][Medline]
Ragland, W. L., Novak, R., El-Attrache, J., Savic, V. & Ester, K. (2002). Chicken anemia virus and infectious bursal disease virus interfere with transcription of chicken IFN- and IFN-
mRNA. J Interferon Cytokine Res 22, 437441.[CrossRef][Medline]
Rosell, C., Segales, J., Ramos-Vara, J. A., Folch, J. M., Rodriguez-Arrioja, G. M., Duran, C. O., Balasch, M., Plana-Duran, J. & Domingo, M. (2000). Identification of porcine circovirus in tissues of pigs with porcine dermatitis and nephropathy syndrome. Vet Rec 146, 4043.[Medline]
Rosenberger, J. K. & Cloud, S. S. (1998). Chicken anemia virus. Poult Sci 77, 11901192.[Medline]
Sandvik, T., Grierson, S., King, D., Spencer, Y., Banks, M. & Drew, T. (2001). Detection and genetic typing of porcine circovirus DNA isolated from archived paraffin embedded pig tissues. ssDNA Viruses of Plants, Birds, Pigs and Primates (Saint-Malo, France, 2427 September 2001).
Sato, Y., Roman, M., Tighe, H. & 7 other authors (1996). Immunostimulatory DNA sequences necessary for effective intradermal gene immunization. Science 273, 352354.[Abstract]
Sato, Y., Miyata, M., Sato, Y., Nishimaki, T., Kochi, H. & Kasukawa, R. (1999). CpG motif-containing DNA fragments from sera of patients with systemic lupus erythematosus proliferate mononuclear cells in vitro. J Rheumatol 26, 294301.[Medline]
Stein, C. A., Subasinghe, C., Shinozuka, K. & Cohen, J. S. (1988). Physicochemical properties of phosphorothioate oligodeoxynucleotides. Nucleic Acids Res 16, 32093221.[Abstract]
Stevenson, G. W., Kiupel, M., Mittal, S. K., Choi, J., Latimer, K. S. & Kanitz, C. L. (2001). Tissue distribution and genetic typing of porcine circoviruses in pigs with naturally occurring congenital tremors. J Vet Diagn Invest 13, 5762.[Medline]
Stunz, L. L., Lenert, P., Peckham, D., Yi, A. K., Haxhinasto, S., Chang, M., Krieg, A. M. & Ashman, R. F. (2002). Inhibitory oligonucleotides specifically block effects of stimulatory CpG oligonucleotides in B cells. Eur J Immunol 32, 12121222.[CrossRef][Medline]
Sun, S., Beard, C., Jaenisch, R., Jones, P. & Sprent, J. (1997). Mitogenicity of DNA from different organisms for murine B cells. J Immunol 159, 31193125.[Abstract]
Tighe, H., Corr, M., Roman, M. & Raz, E. (1998). Gene vaccination: plasmid DNA is more than just a blueprint. Immunol Today 19, 8997.[CrossRef][Medline]
Tischer, I., Rasch, R. & Tochtermann, G. (1974). Characterization of papovavirus- and picornavirus-like particles in permanent pig kidney cell lines. Zentralbl Bakteriol 226, 153167.
Tischer, I., Mields, W., Wolff, D., Vagt, M. & Griem, W. (1986). Studies on epidemiology and pathogenicity of porcine circovirus. Arch Virol 91, 271276.[Medline]
Vallin, H., Perers, A., Alm, G. V. & Ronnblom, L. (1999). Anti-double-stranded DNA antibodies and immunostimulatory plasmid DNA in combination mimic the endogenous IFN- inducer in systemic lupus erythematosus. J Immunol 163, 63066313.
Verthelyi, D., Ishii, K. J., Gursel, M., Takeshita, F. & Klinman, D. M. (2001). Human peripheral blood cells differentially recognize and respond to two distinct CPG motifs. J Immunol 166, 23722377.
Walker, I. W., Konoby, C. A., Jewhurst, V. A., McNair, I., McNeilly, F., Meehan, B. M., Cottrell, T. S., Ellis, J. A. & Allan, G. M. (2000). Development and application of a competitive enzyme-linked immunosorbent assay for the detection of serum antibodies to porcine circovirus type 2. J Vet Diagn Invest 12, 400405.[Medline]
Wattrang, E., McNeilly, F., Allan, G. M., Greko, C., Fossum, C. & Wallgren, P. (2002). Exudative epidermitis and porcine circovirus-2 infection in a Swedish SPF-herd. Vet Microbiol 86, 281293.[CrossRef][Medline]
Xu, Y. & Szoka, F. C., Jr (1996). Mechanism of DNA release from cationic liposome/DNA complexes used in cell transfection. Biochemistry 35, 56165623.[CrossRef][Medline]
Yamada, H., Gursel, I., Takeshita, F., Conover, J., Ishii, K. J., Gursel, M., Takeshita, S. & Klinman, D. M. (2002). Effect of suppressive DNA on CpG-induced immune activation. J Immunol 169, 55905594.
Yamamoto, S., Yamamoto, T., Shimada, S., Kuramoto, E., Yano, O., Kataoka, T. & Tokunaga, T. (1992). DNA from bacteria, but not from vertebrates, induces interferons, activates natural killer cells and inhibits tumor growth. Microbiol Immunol 36, 983997.[Medline]
Zhao, H., Cheng, S. H. & Yew, N. S. (2000). Requirements for effective inhibition of immunostimulatory CpG motifs by neutralizing motifs. Antisense Nucleic Acid Drug Dev 10, 381389.[Medline]
Received 21 May 2003;
accepted 23 July 2003.
HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
INT J SYST EVOL MICROBIOL | MICROBIOLOGY | J GEN VIROL |
J MED MICROBIOL | ALL SGM JOURNALS |