Porcine Circovirus Type 2 Replication Research Articles

Porcine circovirus type 2 (PCV2) Cap and Rep proteins are involved in the development of cell-mediated immunity upon PCV2 infection

The aim of the present study was to investigate the role of the capside (Cap) and replicase (Rep) proteins of Porcine circovirus type 2 (PCV2) as well as the whole PCV2 (both PCV2a and PCV2b genotypes) in the induction of cell-mediated immunity upon infection. At 6 weeks of age, six pigs were intranasally inoculated with the Stoon 1010 (Stoon) isolate (PCV2a) and seven with the Sp-7-10-54-13 (Sp) isolate (PCV2b). None of the pigs developed clinical disease but the Sp group had significantly higher proportion of pigs with PCV2-associated lesions and PCV2 load in tissues compared to the Stoon group. In both groups, development of IFN-γ secreting cells (SC) in response to the whole PCV2 and Cap protein was detected by means of an ELISPOT from day 7 post-inoculation (PI) to the end of the study (21 days PI). Significant responses against Rep protein were only detected in Sp-inoculated pigs. No differences in ELISPOT results were seen when either PCV2a or PCV2b was used in vitro to recall peripheral blood mononuclear cells (PBMC) in any group. Stimulation of PBMC with the whole virus but not with Cap or Rep protein induced IL-10-SC in all pigs regardless of their PCV2 infection status, indicating an innate origin of this response. The results from this study demonstrate that PCV2-infected pigs developed cell-mediated immunity to Cap and Rep proteins and that, in the course of a sub-clinical infection, development and strength of such responses are possibly related to the levels of PCV2 replication.

Subcellular Immunolocalization of Porcine Circovirus Type 2 (PCV2) in Lymph Nodes from Pigs with Post-weaning Multisystemic Wasting Syndrome (PMWS)

Post-weaning multisystemic wasting syndrome (PMWS) is one of the most significant porcine diseases worldwide. The causative agent is porcine circovirus type 2 (PCV2), the smallest virus known to infect animals. Data related to the structural and ultrastructural aspects of this infectious disease are sparse and there is little knowledge of the subcellular localization of PCV2 and its replication in the tissues of pigs naturally affected by PMWS. The present study describes the cellular localization of PCV2 in the lymph nodes of pigs affected by PMWS by application of immunolabelling techniques for light and transmission electron microscopy (TEM). PCV2 particles were exclusively detected in histiocytes. Ultrastructural alterations including marked dilatation of rough endoplasmic reticulum and swelling of mitochondria were associated with PCV2-labelled intracytoplasmic inclusions (ICIs) with recognizable virions. Within the ICIs icosahedral virus-like particles were specifically labelled with a PCV2 capsid antibody, whereas particles with a granular appearance were not labelled. Colocalization studies with confocal microscopy and double immunolabelling with TEM indicated a close relationship between virus and the mitochondria, suggesting that these organelles may play an important role in the replication of PCV2. The present findings further support the hypothesis that virus replicates within the histiocytes of lymph nodes.

The open reading frame 3 (ORF3) of porcine circovirus type 2 (PCV2) is dispensable for virus infection but evidence of reduced pathogenicity is limited in pigs infected by an ORF3-null PCV2 mutant

Porcine circovirus type 2 (PCV2) is the primary causative agent of porcine circovirus-associated diseases (PCVAD) in pigs. The open reading frame (ORF) 3 of PCV2 reportedly induces apoptosis and is associated with PCV2 pathogenicity. In this study, we first created an ORF3-null PCV2 mutant (muPCV2) by site-directed mutagenesis and demonstrated that the dimerized plasmid DNA of muPCV2 clone is infectious when injected intramuscularly (I.M.) into pigs. Subsequently, by using a well-characterized pig model we compared the pathogenicity of the muPCV2 and the wildtype PCV2. Thirty-one pigs were divided into 3 groups of 11, 10, and 10 each: group 1 pigs were each inoculated I.M. with PBS buffer as negative controls, group 2 pigs each with 200 μg of muPCV2 infectious DNA clone, and group 3 pigs each with 200 μg of wildtype PCV2 infectious DNA clone. Blood was collected prior to inoculation and weekly thereafter, and tested for PCV2 antibodies by ELISA and serum viral DNA loads by quantitative PCR. All pigs were necropsied at 35 days post-inoculation. The results showed that pigs inoculated with muPCV2 had a delayed seroconversion and lower serum viral load. However, there was no significant difference in the average scores of the histological or gross lesions or the amount of PCV2-specific antigen in tissues between wildtype PCV2- and muPCV2-inoculated groups. Thus, the data from this study do not fully support the conclusion of a previous report regarding PCV2 attenuation by abrogation of ORF3 although the results did show that ORF3 is dispensable for PCV2 replication in pigs.

Differential Proteome Analysis of Host Cells Infected with Porcine Circovirus Type 2

Porcine circovirus type 2 (PCV2) is the primary causative agent of postweaning multisystemic wasting syndrome, which is an emerging swine immunosuppressive disease. To uncover cellular protein responses in PCV2-infected PK-15 cells, the comprehensive proteome profiles were analyzed utilizing two-dimensional gel electrophoresis (2-DE) coupled with MALDI-TOF/TOF identification. Multiple comparisons of 2-DE revealed that the majority of changes in protein expression occurred at 48-96 h after PCV2 infection. A total of 34 host-encoded proteins, including 15 up-regulated and 19 down-regulated proteins, were identified by MALDI-TOF/TOF analysis. According to cellular function, the differential expression proteins could be sorted into several groups: cytoskeleton proteins, stress response, macromolecular biosynthesis, energy metabolism, ubiquitin-proteasome pathway, signal transduction, gene regulation. Western blot analysis demonstrated the changes of alpha tubulin, beta actin, and cytokeratin 8 during infection. Colocalization and coimmunoprecipitation analyses confirmed that the cellular alpha tubulin interacts with the Cap protein of PCV2 in the infected PK-15 cells. These identified cellular constituents have important implications for understanding the host interactions with PCV2 and brings us a step closer to defining the cellular requirements for the underlying mechanism of PCV2 replication and pathogenesis.

Interferon-mediated enhancement of in vitro replication of porcine circovirus type 2 is influenced by an interferon-stimulated response element in the PCV2 genome

Porcine circovirus type 2 (PCV2) is an economically important swine pathogen. It has been shown that treatment of PCV2-infected cells with interferon gamma (INF-γ) or alpha (IFN-α) increases PCV2 replication in vitro, and that an interferon-stimulated response element (ISRE)-like sequence was identified in the PCV2 genome. To determine if the ISRE is involved in viral response to IFNs, several ISRE mutants of PCV2 were created by serial mutations of the ISRE sequence. Treatment of ISRE mutants-infected cells with IFN-γ or IFN-α showed a progressive diminishment in the enhanced viral replication in correlation with increasing alterations to the ISRE sequence. To determine if the ISRE was necessary and sufficient for IFN-mediated enhancement of PCV2 replication, DNA fragments spanning the ISRE-containing promoter region of the rep gene from wildtype and ISRE-mutant PCV2 were tested in a luciferase reporter gene system. 3D4/31 cells transfected with reporter gene constructs were treated with IFN-γ or IFN-α, respectively. The results showed that the untreated controls for both ISRE-mutant and wildtype PCV2 had higher levels of luciferase reporter activity than IFN-treated samples, indicating that, when removed from the context of whole viral genome, the ISRE-like activity of the sequence was lost. Furthermore, treatment with IFNs diminished the promoter activity regardless of the mutation, suggesting that other cis elements in the viral genome may be required for regulating the ISRE-mediated gene transcription. In conclusion, the PCV2 ISRE, when present in the context of intact virus but not in isolation, influences the interferon-mediated enhancement of PCV2 replication in vitro.

Effect of Porcine Circovirus Type 2 (PCV2) Vaccination of the Dam on PCV2 Replication In Utero

The aims of this study were to determine if porcine circovirus type 2 (PCV2) vaccination of the dam is effective in preventing fetal PCV2 infection and reproductive failure. Twelve pregnant, PCV2-naïve sows were randomly divided into four groups, with three sows in each group. Group 1 sows served as noninoculated, nonvaccinated negative controls, group 2 sows were vaccinated with a commercially available PCV2 vaccine at 28 days of gestation and were not inoculated, group 3 sows were vaccinated at 28 days of gestation and inoculated with PCV2b at 56 days of gestation, and group 4 sows were inoculated with PCV2b but were not vaccinated. Serum samples from all sows were collected weekly throughout the gestation period, and sows were allowed to farrow naturally. At parturition, sow colostrum samples, presuckle serum samples, and tissues from the piglets were collected. Reproductive failure was not observed under the study conditions. PCV2 vaccination induced PCV2-specific immunoglobulin G and serum neutralizing antibodies in sows from groups 2 and 3 and prevented detectable PCV2 viremia in the dams after challenge. In group 3, PCV2 DNA was detected in colostrum samples, fetuses, and live-born pigs; however, microscopic lesions and PCV2-specific antigen were not present in any of the fetuses in this group. The results from this study indicate that vertical transmission of PCV2 can occur in PCV2-vaccinated dams.

JNK and p38 Mitogen-Activated Protein Kinase Pathways Contribute to Porcine Circovirus Type 2 Infection

Infection with a wide variety of viruses often perturbs host cell signaling pathways including the Jun NH(2)-terminal kinase/stress-activated kinase (JNK/SAPK) and the p38 mitogen-activated protein kinase (p38/MAPK), which are important components of cellular signal transduction pathways. The present study demonstrated for the first time that porcine circovirus type 2 (PCV2), which is the primary causative agent of an emerging swine disease, postweaning multisystemic wasting syndrome, can activate JNK1/2 and p38 MAPK pathways in PCV2-infected PK15 cells. However, PCV2 at an early stage of infection, as well as UV-irradiated PCV2, failed to activate these two MAPK families, which demonstrated that PCV2 replication was necessary for their activation. We further found that PCV2 activated the phosphorylation of JNK1/2 and p38 MAPK downstream targets c-Jun and ATF-2 with virus replication in the cultured cells. The roles of these kinases in PCV2 infection were further evaluated using specific inhibitors: the JNK inhibitor 1 for JNK1/2 and SB202190 for p38. Inhibition of JNK1/2 and p38 kinases by these specific inhibitors did result in significant reduction of PCV2 viral mRNA transcription and protein synthesis, viral progeny release, and blockage of PCV2-induced apoptotic caspase-3 activation in the infected cells. Taken together, these data suggest that JNK/SAPK and p38 MAPK pathways play important roles in the PCV2 replication and contribute to virus-mediated changes in host cells.

Porcine circovirus type 2 replication is impaired by inhibition of the extracellular signal-regulated kinase (ERK) signaling pathway

Postweaning multisystemic wasting syndrome, which is primarily caused by porcine circovirus type 2 (PCV2), is an emerging and important swine disease. We have recently shown that PCV2 induces nuclear factor kappa B activation and its activation is required for active replication, but the other cellular factors involved in PCV2 replication are not well defined. The extracellular signal-regulated kinase (ERK) which served as an important component of cellular signal transduction pathways has been shown to regulate many viral infections. In this report, we show that PCV2 activates ERK1/2 in PCV2-infected PK15 cells dependent on viral replication. The PCV2-induced ERK1/2 leads to phosphorylation of the ternary complex factor Elk-1, which kinetically paralleled ERK1/2 activation. Inhibition of ERK activation with U0126, a specific MEK1/2 inhibitor, significantly reduced viral progeny release. Investigations into the mechanism of ERK1/2 regulation revealed that inhibition of ERK activation leads to decreased viral transcription and lower virus protein expression. These data indicate that the ERK signaling pathway is involved in PCV2 infection and beneficial to PCV2 replication in the cultured cells.

Effects of a modified live CSFV vaccine on the development of PMWS in pigs infected experimentally with PCV-2

The objective of this study was to determine the effect of vaccination against classical swine fever virus (CSFV) on the development of postweaning multisystemic wasting syndrome (PMWS) in conventional pigs...

Effect of porcine circovirus type 2 infection and replication on activated porcine peripheral blood mononuclear cells in vitro

The goal of this study was to examine the effect of porcine circovirus type 2 (PCV2) infection and replication on peripheral blood mononuclear cells (PBMCs) in the presence of mitogens, concanavalin A (ConA) or pokeweed mitogen (PWM) in vitro. The level of PCV2 replication and the impact of infection on PBMC proliferation, viability and the level of apoptosis in the presence or absence of mitogen stimulation were assessed. Mitogen stimulation increased viral replication in PBMCs as measured by the amount of spliced capsid mRNA (Cap mRNA). However, cell proliferation alone had no significant impact on PCV2 replication as the level of Cap mRNA in ConA or PWM stimulated PBMCs was not increased in proliferating cells compared to non-proliferating cells. No significant differences were observed in the level of PCV2 replication product in PBMCs stimulated with ConA for 12, 36, and 72 h prior to infection. Infection with PCV2 did not affect the ability of PBMCs to proliferate in response to ConA or PWM stimulation in vitro. Increased apoptosis was associated with PCV2 infection in PWM stimulated PBMCs. Interestingly, a significantly lower apoptotic index was observed in PCV2 infected PBMCs compared to mock-infected cells in the absence of mitogens. This study determined that the rate of PCV2 replication increases with cell stimulation and apoptosis is increased following PCV2 infection under certain stimulation conditions. These results further suggest that PCV2 requires a specific stimulation or trigger for increased viral replication, which is independent of cell proliferation.

Porcine circovirus type 2 displays pluripotency in cell targeting

Porcine circovirus type 2 (PCV2) is the causative agent of a multifactorial disease associated with immunocompromisation and co-infections. In vivo, viral DNA and antigens are found in monocytic, epithelial and endothelial cells. Of these, PCV2 replication has only been studied in monocytic cells, in which little or no replication was identified. Accordingly, PCV2 infection was studied in the endothelial cell line PEDSV.15, aortic endothelial cells, gut epithelial cells, fibrocytes and dendritic cells (DC). In all cells except DC PCV2 replication was detectable, with an increase in the levels of capsid and replicase protein. Variations in endocytic activity, virus binding and uptake did not relate to the replication efficiency in a particular cell. Furthermore, replication did not correlate to cell proliferation, although a close association of viral proteins with chromatin in dividing cells was observed. No alteration in the division rate of PCV2-infected cultures was measurable, relating to replicase expression in only a small minority of the cells. In conclusion, the broad cell targeting of PCV2 offers an explanation for its widespread tissue distribution.

Rolling-circle amplification for the detection of active porcine circovirus type 2 DNA replication in vitro

Porcine circovirus type 2 (PCV2) infections in pigs have diverse clinical presentations and are considered economically important diseases worldwide. However, despite intensive research, the early pathogenesis of PCV2 and the primary target cells for PCV2 infections and replication are still unknown. Rolling-circle amplification (RCA) is an amplification technique for small, circular DNA templates that essentially mimics rolling-circle replication in vitro. In this study, the amplification of PCV2-specific DNAs using randomly primed RCA has been demonstrated. This novel approach has circumvented the normal requirement for conventional virus isolation procedures for the characterization of PCV2 DNAs from clinical samples. In addition, the potential utility of a strand-specific derivative of RCA was further investigated. Specifically, strand-specific RCA for the detection of active virus replication following the amplification of complementary sense PCV2 DNAs, which occur as double-stranded replicative intermediates that are present only during de novo viral DNA replication both in vitro and in vivo has been demonstrated.

Porcine circovirus type 2 induces the activation of nuclear factor kappa B by IκBα degradation

The transcription factor NF-κB is commonly activated upon virus infection and a key player in the induction and regulation of the host immune response. The present study demonstrated for the first time that porcine circovirus type 2 (PCV2), which is the primary causative agent of an emerging swine disease, postweaning multisystemic wasting syndrome, can activate NF-κB in PCV2-infected PK15 cells. In PCV2-infected cells, NF-κB was activated concomitantly with viral replication, which was characterized by increased DNA binding activity, translocation of NF-κB p65 from the cytoplasm to the nucleus, as well as degradation and phosphorylation of IκBα protein. We further demonstrated PCV2-induced activation of NF-κB and colocalization of p65 nuclear translocation with virus replication in cultured cells. Treatment of cells with CAPE, a selective inhibitor of NF-κB activation, reduced virus protein expression and progeny production followed by decreasing PCV2-induced apoptotic caspase activity, indicating the involvement of this transcription factor in induction of cell death. Taken together, these data suggest that NF-κB activation is important for PCV2 replication and contributes to virus-mediated changes in host cells. The results presented here provide a basis for understanding molecular mechanism of PCV2 infection.

Increased porcine circovirus type 2 replication in porcine leukocytes in vitro and in vivo by concanavalin A stimulation

Previously, it was shown that modulation of the immune system enhances porcine circovirus type 2 (PCV2) replication in pigs. In the present study, the effect of the mitogen concanavalin A (ConA) on PCV2 replication was investigated. Since ConA induces T-lymphocyte activation and initiates the production of interferon-gamma (IFN-γ), a cytokine that enhances PCV2 replication in porcine epithelial and monocytic cell lines in vitro, it was examined if the effects observed with ConA were mediated by IFN-γ. In an in vitro study, ConA but not IFN-γ enhanced PCV2 replication in peripheral blood mononuclear cells (PBMC). Up to 2.08% and 0.96% of PBMC were antigen positive for PCV2 strains 1121 and Stoon-1010, respectively, and a low virus production was observed. PCV2-infected PBMC were identified as CD4 + (40%), CD8 + (54%) and IgM + (11%). In a subsequent in vivo study, caesarean-derived colostrum-deprived piglets were injected with ConA or IFN-γ 12 h before inoculation and every 3 days for 9 days after inoculation with strain 1121. PCV2 was isolated from inguinal lymph node biopsies from 10 days post-inoculation (dpi) in ConA-treated pigs and from 15 dpi in non-treated and IFN-γ-treated pigs. ConA increased PCV2 replication levels, but disease was not observed. Half of the ConA-treated and IFN-γ-treated pigs showed a delayed humoral immune response, but this delay did not result in increased PCV2 replication in these pigs. These experiments demonstrated that ConA enhances PCV2 replication in PBMC in vitro and in lymphoid tissues in vivo.

Effect of different selenium sources and levels on porcine circovirus type 2 replication in vitro

Porcine circovirus type 2 (PCV2) has been linked to several disease syndromes during the last decade. A deficiency in selenium has also been associated with the increases of virulence of some viruses and severity of infectious disease. In order to evaluate the effect of different selenium sources and levels on PCV2 replication in PK-15 cells, three selenium sources, i.e. sodium selenite, kappa-selenocarrageenan and dl-selenomethionine at concentrations of 0, 2, 4, 8, and 16 μmol/L were used throughout this experiment. PCV2 loads in PK-15 cells were measured by a newly developed real-time quantitative PCR. A significantly inhibitive effect of dl-selenomethionine on PCV2 replication in vitro was demonstrated and the inhibition was concentration dependent within the range of 2–16 μmol/L. The inhibitive effect of dl-selenomethionine on PCV2 replication may be caused by enhanced activity of glutathione peroxidase. Our results may serve as a basis for further studies of the biological function of selenium and control of PCV2 infection.

Adenovirus-mediated shRNA interference against porcine circovirus type 2 replication both in vitro and in vivo

Porcine circovirus type 2 (PCV2) is the primary causative agent of an emerging swine disease, postweaning multisystemic wasting syndrome (PMWS), which is responsible for the heavy economic losses in stockbreeding. There are no specific antiviral drugs for treatment of the virus infection. We have now constructed two recombinant adenoviruses expressing short-hairpin RNAs (shRNAs) directed against either ORF1 (rAdS1) or ORF2 (rAdS2) of PCV2 and measured the inhibition of PCV2 replication. The results showed that delivery of these shRNAs by recombinant adenovirus into PK15 cells could induce a significant inhibition of viral RNA and DNA replication and protein synthesis level in cells subsequently infected with PCV2. The antiviral effect was dose-dependent and could sustain at least for 120 h and the inhibition of virus replication could be significantly strengthened by combination of rAdS1 with rAdS2. Mice injected with shRNA before PCV2 infection showed substantial and low level of PCV2 DNA replication in the spleen during the period of 21–28 days post-PCV2 infection. These results indicated that shRNAs generated by adenovirus could sufficiently and continuously inhibit PCV2 infection in vitro as well as in vivo. The adenovirus based shRNA targeting ORF1 and ORF2 of PCV2 might be a new potential alternative strategy for controlling PCV2 infection.

Lack ofIn VitroandIn VivoEffects of Lipopolysaccharide on Porcine Circovirus Type 2 Infection

Porcine circovirus type 2 (PCV2) is the causative agent of postweaning multisystemic wasting syndrome (PMWS). The presence of immunostimulating factors or concurrent infections seems to be crucial for PMWS development. Lipopolysaccharide (LPS) is a potent immunological activator and has recently been suggested to enhance PCV2 replication in vitro. This study was designed to evaluate the effects of different LPS products on PCV2 in vitro replication of pulmonary macrophages (PMs), and on the potential ability to trigger PMWS in cesarean-derived, colostrum-deprived (CDCD) PCV2-inoculated piglets. In vitro studies using two different PCV2 isolates (Stoon-1010 and 1452/3) showed the presence of PCV2 antigen within the cytoplasm to a variable degree; PCV2 Stoon-1010 was barely detectable (<1% of stained cells), and PCV2 1452/3 was seen in the cytoplasm of more than 85% of PMs. However, no differences were found in intracytoplasmic PCV2 signals among different LPS treatments, or between the LPS-treated and non-treated PMs. Moreover, almost no intranuclear signals for PCV2 antigen were detected in PMs. The in vivo experiment included twenty 7-day-old CDCD piglets divided into four groups: control (n = 4), control/LPS (n = 4), PCV2 (n = 6), and PCV2/LPS (n = 6). The control and control/LPS groups were inoculated intranasally with a cell culture medium (MEM), and the PCV2 and PCV2/LPS groups were inoculated with a Spanish isolate of PCV2 (Burgos). The control/LPS and PCV2/LPS groups were inoculated intraperitoneally with LPS on PCV2 inoculation day. All pigs remained clinically healthy during the entire experimental period (29 days). Animals inoculated with LPS had significant hyperthermia within the first 24 hours post-inoculation. No differences in gross or histological findings were observed among the PCV2 and PCV2/LPS inoculated pigs. All PCV2-infected piglets developed a subclinical infection with the virus. Our results showed that LPS did not increase in vitro viral replication and did not trigger PMWS in PCV2-inoculated pigs.

Enhanced replication of porcine circovirus type 2 (PCV2) in a homogeneous subpopulation of PK15 cell line

Post-weaning multi-systemic wasting syndrome (PMWS) has emerged as a major disease that poses a significant threat to the economics of global swine industry. Porcine circovirus type 2 (PCV2) is the causal agent of PMWS in pigs. Currently, the prevention of PCV2 infection based on vaccines is limited, and the available vaccines are either killed viral vaccines or recombinant protein based vaccines and not cost effective. The PK-15 cells, which is widely used for PCV2 propagation, is not efficient and heterogeneous in terms of permissivity to viral infection. In order to acquire a homogeneous porcine kidney cell line that can reliably produce PCV2 in high titers, cell clones that show high- (PK15-C1) or low-permissive (PK15-A2) phenotype to PCV2 infection were derived from heterogeneous PK15 parent cells by limiting dilution and cell cloning. Maximum virus titers in PK15-C1, PK15-A2 and PK15 parent cells were 10 8, 10 2 and 10 5 tissue culture infective dose 50 (TCID 50)/ml, respectively. The viral proteins of PCV2 were produced and accumulated faster in PK15-C1 cells than those in PK15 parent cells. These results indicate that PK15-C1 cell clone is more permissive to PCV2 infection than PK15 parent cells and thus will be useful for PCV2 replication in vitro, as well as, vaccines, diagnostic and research applications on PCV2.

Immunogenicity of porcine circovirus type 2 capsid protein targeting to different subcellular compartments

Porcine circovirus type 2 (PCV2) is known to be associated with post-weaning multisystemic wasting syndrome (PMWS), an emerging disease in swine. The development of effective vaccines against PCV2 infection has been accepted as an important strategy in the prophylaxis of PMWS, and a DNA vaccine expressing the major immunogenic capsid (Cap) protein of PCV2 is considered to be a promising candidate. However, recent studies have revealed that interferons (IFNs), especially IFN-γ, can enhance the replication of PCV2, indicating that the high levels of IFN-γ induced by DNA vaccination seem to have potential deleterious effect on protective immunity. Strategies to improve the neutralizing antibody response and simultaneously decrease the IFN-γ response will facilitate the clinical application of DNA vaccines against PCV2. In the present study, four different DNA vaccine constructs encoding cytoplasmic (Cy-ORF2), secreted (Sc-ORF2), membrane-anchored (M-ORF2) or authentic nuclear-targeted (pc-ORF2) Cap protein were generated to evaluate the neutralizing antibody and IFN-γ responses in a mouse model. Although all four DNA constructs could elicit PCV2-specific humoral immune responses, mice inoculated with Sc-ORF2 developed a significantly higher level of neutralizing antibodies than those that received M-ORF2, pc-ORF2 or Cy-ORF2. Furthermore, mice immunized with Sc-ORF2 or M-ORF2 showed a significantly decreased or enhanced IFN-γ level, respectively, compared with those inoculated with pc-ORF2. With respect to neutralizing antibody and IFN-γ levels, Sc-ORF2 is a good candidate for DNA vaccination, and the secreted Cap protein appears to be an ideal antigen for use in development of vaccines against PCV2.

A new method to identify cell types that support porcine circovirus type 2 replication in formalin-fixed, paraffin-embedded swine tissues

Porcine circovirus type 2 (PCV2) is detected in high amounts within the characteristic microscopic lesions of postweaning multisystemic wasting syndrome (PMWS) affected pigs. In spite of recent advances on disease pathogenesis, the precise cell types that support viral replication are still a major issue of scientific discussion. In this study, a new methodology to detect cell types that support PCV2 replication was designed. For this purpose, two in situ hybridisation (ISH) methods were developed and applied on tissues of PMWS naturally affected pigs using two probes designed from the ORF1 sequence of the virus. While the complementary probe (CP) detected ssDNA, ORF1 mRNA and replicative form (RF) of PCV2, the RF probe (RFP) exclusively hybridised with the RF of the virus, thus, only labelling cells where PCV2 replication is taking place. Both probes demonstrated to be specific and equally sensitive by an in vitro Southern blot hybridisation assay. ISH labelling with the CP was extensive in lymphoid tissues and of variable amount in other non-lymphoid tissues. With this probe, mainly macrophage-like cells were labelled but also other cell types such as hepatocytes and other epithelial cells. Tissues in which RFP labelling was found more frequently were lung, inguinal and mesenteric lymph nodes, tonsil and liver. Labelling with the RFP was always nuclear, and found in the same cell types as with the CP, although in a relatively low proportion of them; labelling of macrophage-like cells was infrequent. Therefore, the results indicate that at least a certain proportion of macrophages may support PCV2 replication, but main cells where PCV2 replicates are of epithelial/endothelial origin. In summary, the present study permitted the study of cell types that support PCV2 replication by the use of ISH on formalin-fixed, paraffin-embedded tissues of PMWS affected pigs.