Porcine Circovirus Type 2 Group Research Articles

Importance of sequential infection order of porcine circovirus type 2 and porcine reproductive and respiratory syndrome virus to divergent clinical outcomes

This study was designed to investigate the different sequential order of infection for porcine circovirus type 2 (PCV2) and porcine reproductive and respiratory syndrome virus (PRRSV). Thirty-six pigs were randomly assigned to six different treatment groups. The first (hereafter referred to as PRRSV-PCV2) group was inoculated with PRRSV first followed by PCV2d. The second (hereafter referred to as PCV2+PRRSV) group was co-infected with both viruses at the same timepoint (42 days of age). The third (hereafter referred to as PCV2-PRRSV) group was inoculated with PCV2d first followed by PRRSV. A fourth group was only inoculated with PCV2d at 42 days of age, while a fifth group was only inoculated with PRRSV at the same timepoint. The sixth group served as a negative control group. The most important observation discovered that PRRSV only had a potentiation effect on PCV2 in both PRRS-PCV2 and PCV2+PRRSV groups. Both PRRSV-PCV2 and PCV2+PRRSV groups experienced a significant reduction in growth performance compared with control pigs. In addition, PRRSV-PCV2 and PCV2+PRRSV groups exhibited a greater severity in their clinical signs, and/or had higher PCV2 blood and lymphoid viral loads that resulted in a stronger severity of lymphoid lesions compared with PCV2-PRRSV group. Serum TNF-α levels were significantly higher in both PRRS-PCV2 and PCV2+PRRSV groups compared with those in PCV2-PRRS, PCV2, and PRRSV groups. The results of this study demonstrated that divergent clinical outcomes are dependent on the sequential infection order of PCV2 and PRRSV.

Experimental infection of high health pigs with porcine circovirus type 2 (PCV2) and Lawsonia intracellularis.

BackgroundPorcine circovirus type 2 (PCV2) and Lawsonia intracellularis infections can cause enteritis in pigs. A Danish study showed a significantly higher probability of detecting PCV2 without concurrent L. intracellularis infection, indicating that one of these pathogens has an impact on the dynamics of the other. Therefore, a delayed co-infection model was set up, initially aiming at investigating the interaction between PCV2 and L. intracellularis in pigs challenged with PCV2 and 2 weeks later with L. intracellularis. But due to PCV2 contamination of the L. intracellularis inoculum the aim was revisited to describing the infection dynamics and pathogenesis of pigs infected with PCV2 followed by delayed simultaneous exposure to PCV2 and L. intracellularis. Twenty-four high-health piglets were divided into three groups of eight pigs (A, B, C) and inoculated at experimental day (EXD) 0 with mock (groups A and B) or PCV2 (group C), and at EXD 14 with mock (group A) or L. intracellularis/PCV2 (groups B and C). The pigs underwent daily clinical examination, and were necropsied at EXD 51–52. Furthermore, histology, immunohistochemistry, serology and PCR for PCV2 and L. intracellularis, and measurement of C-reactive protein were carried out.ResultsGroup A remained negative for PCV2 and L. intracellularis. Following inoculation with L. intracellularis/PCV2, no significant differences were observed between group B and C, however pigs already infected with PCV2 (group C) showed milder clinical signs and exhibited milder intestinal lesions, less shedding of L. intracellularis and developed higher L. intracellularis antibody titers than the pigs in group B that only received the combined infection. Though the differences between group B and C were non-significant, all results pointed in the same direction, indicating that the pigs in group B were more affected by the L. intracellularis infection compared to the pigs in group C.ConclusionsPrevious exposure to PCV2 had limited impact on the subsequent exposure to a combined L. intracellularis/PCV2 inoculation. However, there was a tendency that the infection dynamics of PCV2 and development of antibodies to PCV2 and L. intracellularis were altered in pigs previously exposed to PCV2. These differences should be confirmed in further experimental trials.

CGAS/STING signaling pathways induces the secretion of type Ⅰ interferon in porcine alveolar macrophages infected with porcine circovirus type 2

In order to study the signal pathway secreting type Ⅰ interferon in porcine alveolar macrophages (PAMs) infected with porcine circovirus type 2 (PCV2), the protein and the mRNA expression levels of cGAS/STING pathways were analyzed by ELISA, Western blotting and quantitative reverse transcriptase PCR in PAMs infected with PCV2. In addition, the roles of cGAS, STING, TBK1 and NF-κB/P65 in the generation of type I interferon (IFN-I) from PAMs were analyzed by using the cGAS and STING specific siRNA, inhibitors BX795 and BAY 11-7082. The results showed that the expression levels of IFN-I increased significantly at 48 h after infection with PCV2 (P<0.05), the mRNA expression levels of cGAS increased significantly at 48 h and 72 h after infection (P<0.01), the mRNA expression levels of STING increased significantly at 72 h after infection (P<0.01), and the mRNA expression levels of TBK1 and IRF3 increased at 48 h after infection (P<0.01). The protein expression levels of STING, TBK1 and IRF3 in PAMs infected with PCV2 were increased, the content of NF-κB/p65 was decreased, and the nuclear entry of NF-κB/p65 and IRF3 was promoted. After knocking down cGAS or STING expression by siRNA, the expression level of IFN-I was significantly decreased after PCV2 infection for 48 h (P<0.01). BX795 and BAY 11-7082 inhibitors were used to inhibit the expression of IRF3 and NF-κB, the concentration of IFN-I in BX795-treated group was significantly reduced than that of the PCV2 group (P<0.01), while no significant difference was observed between the BAY 11-7028 group and the PCV2 group. The results showed that PAMs infected with PCV2 induced IFN-I secretion through the cGAS/STING/TBK1/IRF3 signaling pathway.

Porcine Circovirus Type 2 Pathogenicity Alters Host's Central Tolerance for Propagation.

Porcine circovirus type 2 (PCV2) infections and resulting diseases are a worldwide threat to pig production. PCV2 bears a uniqueness that allows for us to understand more about chronic infections and the immune system in general. The virus can be phylogenetically subdivided into PCV2a to PCV2h genotypes. Although vaccination against PCV2 has been seen to prevent the manifestation of PCV disease, PCV2 still lingers as subclinical infections in all developmental stages of pigs. The “slow and low” tactic gives PCV2 a particular advantage in a host’s immune surveillance. Since the inception of the PCV2 associated panzootic, research scientists have been trying to understand the pathogenicity of PCV2. Different research groups found that one genotype group member was more pathogenic than others. We found, in our weaner infection model with in vivo transfection of different recombinant PCV2 genotype group members that these viruses alter T cell maturation in the thymus, including host’s central tolerance. Here, we extend these original observations by showing that PCV2 infected cells were also found in proximity within the female and male reproductive organs of stillborn pig fetuses. These PCV2 pools were sufficient in infecting three and half-day-old embryos in sows. Furthermore, the dominant PCV2 group member was more pathogenic in our weaner infection model. PCV2 pre-immunocompetence infection makes PCV2 recognized by central immune tolerance as belonging to the host. This also explains why pathogenicity is not a genetically intrinsic characteristic of PCV2; however, the dominance of any one PCV2 genotype group member leads to a more efficient deletion of the T cells against that specific genotype group member in the thymus.

Immune responses induced by a combined vaccination with a recombinant chimera of Mycoplasma hyopneumoniae antigens and capsid virus-like particles of porcine circovirus type 2

BackgroundMycoplasma hyopneumoniae (Mhp) and porcine circovirus type 2 (PCV2) are two important pathogens causing Mycoplasma pneumonia of swine (MPS) and porcine circovirus diseases and porcine circovirus-associated diseases (PCVDs/PCVADs), respectively, and resulted in considerable economic loss to the swine industry worldwide. Currently, vaccination is one of the main measures to control these two diseases; however, there are few combination vaccines that can prevent these two diseases. To determine the effect of combination immunization, we developed capsid-derived (Cap) virus-like particles (VLPs) of PCV2 and a new recombinant chimera composed of the P97R1, P46, and P42 antigens of Mhp. Then we investigated the immune responses induced by the immunization with this combination vaccine in mice and piglets.ResultsThe high level antibodies against three protein antigens (P97R1, P46, and P42 of Mhp) were produced after immunization, up to or higher than 1:400,000; the antibody levels in Pro group continuously increased throughout the 42 days for all the antigens tested. The lymphocyte proliferative response in PCV2 group was stronger than that in PBS, VP, Mhp CV in mice. The antibody levels for Cap remained stable and reached the peak at 35 DAI. The IFN-γ and IL-4 in sera were significantly enhanced in the Pro group than that in the negative control-VP group on Day 14 and 28 post-the first immunization in piglets.ConclusionsAbove all, the combination immunization could induce humoral and cellular immune responses against all four antigens in mice and piglets. Therefore, our approach is a simple and effective vaccination strategy to protect pigs against MPS and PCVD/PCVAD.

Effect of porcine circovirus type 2 on the severity of lung and brain damage in piglets infected with porcine pseudorabies virus

Porcine circovirus type 2 (PCV2) is widespread throughout Chinese farms, and the infection rate of porcine pseudorabies virus (PRV) is very high. The emergence of mixed infection involving PCV2 and PRV has been difficult to prevent and control and has caused considerable economic loss. The present study investigated lung and brain damage caused by PRV in piglets with PCV2 infection. Twenty piglets were divided randomly into two experiment groups (PRV group and PRV + PCV2 group; n = 10 per group). The pigs were observed for clinical signs at specified times. At necropsy, lung and brain tissue samples were collected for histopathological examination, and tissue virus load was determined using quantitative polymerase chain reaction. Severe pathogenicity due to PRV was evident in two-month-old piglets. PCV2 and PRV co-infection led to more severe neurological and respiratory symptoms and a higher mortality rate in the piglets. In addition, the pathological damage to the lung and brain was also aggravated. The co-infection was associated with a significant increase in the content of PRV in the brain and lung tissue. In conclusion, PCV2 and PRV co-infection could cause severe and irreversible damage to piglets.

Reduced antigen presentation capability and modified inflammatory/immunosuppressive cytokine expression of induced monocyte-derived dendritic cells from peripheral blood of piglets infected with porcine circovirus type 2.

The efficiency of immune responses and host defense against pathogens largely depends on the function of dendritic cells (DCs). Porcine circovirus type 2 (PCV2) infection causes viremia and extensive modulation of immune activities in the blood. The objective of the present study was to investigate the effects of PCV2 infection in vivo on the immunological function of DCs induced from peripheral blood monocytes (MoDCs). At different points after infection with PCV2, peripheral blood monocytes from PCV2-infected pigs were used to induce differentiation of DCs in vitro. Flow cytometry and quantitative real-time reverse transcription PCR were conducted to detect mRNA expression of surface markers related to antigen presentation and inflammatory/immunosuppressive cytokines of the induced MoDCs. The ability of induced MoDCs to stimulate T cells was measured using an MTS assay. In the early phase of infection at 3 days post-inoculation (DPI), IL-10, IL-8 and MIP-1β in MoDCs were upregulated significantly. By the peak of virus proliferation at 7 DPI, antigen presentation molecules SLA-DR (MHC II) and CD80/86 together with cytokines IL-12 and IL-10 had decreased, accompanied by a rapid reduction of IL-8 and MIP-1β. The T cell stimulation index of induced MoDCs in PCV2 groups after different infection times declined to some extent, with a significant difference at 7 DPI. PCV2 infection in vivo functionally reduced the antigen presentation capability of induced MoDCs from peripheral blood and modified expression of inflammatory/immunosuppressive cytokines that may be related to PCV2-induced immunosuppression.

The Positive Correlation of the Enhanced Immune Response to PCV2 Subunit Vaccine by Conjugation of Chitosan Oligosaccharide with the Deacetylation Degree.

Chitosan oligosaccharides (COS), the degraded products of chitosan, have been demonstrated to have versatile biological functions. In primary studies, it has displayed significant adjuvant effects when mixed with other vaccines. In this study, chitosan oligosaccharides with different deacetylation degrees were prepared and conjugated to porcine circovirus type 2 (PCV2) subunit vaccine to enhance its immunogenicity. The vaccine conjugates were designed by the covalent linkage of COSs to PCV2 molecules and administered to BALB/c mice three times at two-week intervals. The results indicate that, as compared to the PCV2 group, COS–PCV2 conjugates remarkably enhanced both humoral and cellular immunity against PCV2 by promoting lymphocyte proliferation and initiating a mixed T-helper 1 (Th1)/T-helper 2 (Th2) response, including raised levels of PCV2-specific antibodies and an increased production of inflammatory cytokines. Noticeably, with the increasing deacetylation degree, the stronger immune responses to PCV2 were observed in the groups with COS-PCV2 vaccination. In comparison with NACOS (chitin oligosaccharides)–PCV2 and LCOS (chitosan oligosaccharides with low deacetylation degree)–PCV2, HCOS (chitosan oligosaccharides with high deacetylation degree)–PCV2 showed the highest adjuvant effect, even comparable to that of PCV2/ISA206 (a commercialized adjuvant) group. In summary, COS conjugation might be a viable strategy to enhance the immune response to PCV2 subunit vaccine, and the adjuvant effect was positively correlated with the deacetylation degree of COS.

Astragalus polysaccharides attenuate PCV2 infection by inhibiting endoplasmic reticulum stress in vivo and in vitro

This study explored the effects of Astragalus polysaccharide (APS) on porcine circovirus type 2 (PCV2) infections and its mechanism in vivo and vitro. First, fifty 2-week-old mice were randomly divided into five groups: a group without PCV2 infection and groups with PCV2 infections at 0, 100, 200 or 400 mg/kg APS treatments. The trial lasted for 28 days. The results showed that APS treatments at 200 and 400 mg/kg reduced the pathological injury of tissues, inhibited PCV2 infection and decreased glucose-regulated protein 78 (GRP78) and GADD153/CHOP gene mRNA and protein expression significantly (P < 0.05). Second, a study on endoplasmic reticulum stress mechanism was carried out in PK15 cells. APS treatments at 15 and 45 μg/mL significantly reduced PCV2 infection and GRP78 mRNA and protein expression (P < 0.05). Tunicamycin supplementation increased GRP78 mRNA and protein expression and significantly attenuated the APS-induced inhibition of PCV2 infection (P < 0.05). Tauroursodeoxycholic acid supplementation decreased GRP78 mRNA and protein expression and significantly inhibited PCV2 infection (P < 0.05). In addition, fifty 2-week-old mice were randomly divided into five groups: Con, PCV2, APS + PCV2, TM + PCV2 and TM + APS + PCV2. The results were similar to those in PK15 cells. Taken together, it could be concluded that APS suppresses PCV2 infection by inhibiting endoplasmic reticulum stress.

Synergistic pathogenicity in sequential coinfection with Mycoplasma hyorhinis and porcine circovirus type 2

To investigate the synergistic pathogenicity of Mycoplasma hyorhinis (Mhr) with porcine circovirus type 2 (PCV2), thirty 35-day-old piglets were randomly distributed to six groups (n=5 each): Mhr/PCV2 (group 1, inoculated with Mhr and PCV2 1 week later), PCV2/Mhr (group 2, inoculated with PCV2 and Mhr 1 week later), Mhr–PCV2 (group 3, inoculated with PCV2 and Mhr concurrently), singular PCV2 group (group 4), singular Mhr group (group 5), and a uninfected control group (group 6). Mild transient lethargy, fever, coughing, inappetence, and decreased daily weight gain were observed in all dual-infected groups and the singular Mhr-infected group. There were significantly higher levels of PCV2 and Mhr antibodies, larger amounts and wider range of tissue distribution of PCV2 antigens and nucleic acids in the dual-infected groups compared to the single-infected and control groups. PCV2 and Mhr dual-infection resulted in significantly more severe macroscopic and microscopic lung lesions and wider PCV2 DNA distribution compared with piglets infected with PCV2 alone. Cytokine detection showed a significant change in tumor necrosis factor-α, interleukin-2, and interleukin-6 levels in the infected groups, especially in the Mhr–PCV2 group, compared with the control group. Hence, Mhr potentiated the severity of PCV2-associated lung lesions, increased the amount and distribution of PCV2 DNA in tissues, and increased the incidence of porcine respiratory disease.

Population dynamics and ORF3 gene evolution of porcine circovirus type 2 circulating in Korea

This study was conducted to investigate the status and population dynamics of porcine circovirus type 2(PCV2) in Korea and to assess the molecular evolutionary pattern of the two biologically important, overlapping open reading frames, the ORF1 and ORF3 genes. A wide range of PCV2 genomic sequences (entire genome, ORF1, ORF2 and ORF3) collected between 2001 and 2010 were analyzed using the Bayesian Markov chain Monte Carlo and maximum-likelihood approaches. These techniques identified the PCV2d genotype and the 2Ek cluster of PCV2a in Korea for the first time. Second, the genotypic shift of PCV2b dominating over PCV2a likely occurred between 2002 and 2004 due to a population expansion of PCV2b. In the context of positive Darwinian selection, the results uncovered independent evolutionary patterns in the ORF3 gene compared to the overlapping ORF1 gene and new sites in the viral ORFs/proteins that might relate to differences in the biological properties of the PCV2 groups.

Porcine circovirus-associated disease in weaner pigs in Western Australia

To report the occurrence and pathology of porcine circovirus type 2 (PCV2)-associated disease (PCVAD) of postweaning pigs in two Australian pig herds. Mortality data from two commercial piggeries that experienced higher than normal postweaning illthrift and mortalities were examined. Gross and histopathological examinations were performed on the index cases, and at weekly intervals thereafter for a period of 10 weeks. Specimens were submitted to the laboratory for routine diagnostic testing and for exclusion of porcine reproductive and respiratory syndrome virus (PRRSV). The genomes of two strains of PCV2 isolated during testing were sequenced. Mortality rates in weaned, 5-12-week-old pigs spiked significantly during mid to late 2007. This increase in the mortalities was mainly attributed to salmonella-associated diarrhoea and illthrift. Salmonellosis was diagnosed in 73/110 cases inclusive of both piggeries. Many pigs also had chronic granulomatous lymphadenitis and diffuse histiocytic interstitial pneumonia consistent with PCVAD and associated with varying amounts of PCV2 antigen and inclusion bodies. All samples tested for PRRSV were negative. Sequence analysis of the PCV2 isolates showed strain differences between piggeries. This report describes the first outbreaks of PCVAD in growing pigs in Western Australia (WA) and describes lesions not previously seen in this laboratory. It also describes the first isolation of a PCV2 group 1 virus in WA associated with PCVAD. Although the outbreaks of PCVAD occurred with concurrent salmonellosis, the two diseases were unrelated. Neither of the outbreaks met the Australian case definition for the diagnosis of postweaning multisystemic wasting syndrome.

Genetic diversity of porcine circovirus type 2 from pigs in Republic of Korea

Porcine circovirus type 2 (PCV2) is the causative agent of postweaning multisystemic wasting syndrome (PMWS). In this study, 38 PCV2 cases obtained from different pig farms with different health conditions in Republic of Korea were sequenced and analyzed. Phylogenetic analysis showed that our cases had a greater variation and the existence of two PCV2 groups with at least four subgroups (1A, 1C, 2D, and 2E). Most cases obtained from PMWS-affected herds were in group 1, whereas cases with no clinical signs compatible with PMWS (wasted non-PMWS) were included within group 2. Moreover, four cases from the wasted non-PMWS belonged to PCV2-group 1. Therefore, our results suggest that PCV2-group 1 is more related to PMWS than group 2.

Exploratory Study on the Transcriptional Profile of Pigs Subclinically Infected with Porcine Circovirus Type 2

The aim of this exploratory work was to use a microarray-based approach to study the global transcriptome profile of caesarean-derived, colostrum-deprived (CDCD) piglets experimentally infected with porcine circovirus type 2 (PCV2). PCV2-inoculated piglets developed a subclinical infection, as confirmed by serology, in situ hybridization and quantitative PCR. Total RNA from mesenteric lymph nodes and lungs was obtained by duplicate from 2 control and 2 PCV2-inoculated piglets and was hybridized to Affymetrix Porcine GeneChip®. Among the 24,123 probesets studied, 25 and 33 were found to be significantly differentially expressed (DE) between control and PCV2 groups for mesenteric lymph node and lung, respectively. Most up-regulated genes in PCV2 group were closely related to the immune response, such as cytokines (CCL4L, CXCL9, CXCL11), MHC binding molecules (TCRα, CD8α), immunoglubulins (IgG) and T cell activation (LCK, KLRK1, RASSF2, GBP2). Quantitative real-time PCR was performed to verify the microarray results. Therefore, from a transcriptional point of view, PCV2-inoculated pigs were apparently able to activate a cell-mediated response and develop PCV2-specific antibodies, which probably led to a subclinical infection. The results from this study indicate that a microarray based approach is a helpful tool in order to better understand the pathogenesis of PCV2 infection.

Genotypic Change of Porcine Circovirus Type 2 on Japanese Pig Farms as Revealed by Restriction Fragment Length Polymorphism Analysis

Porcine circovirus type 2 (PCV2) has been recognized as the causal agent of postweaning multisystemic wasting syndrome and can be divided into two major genotypic groups. We developed a method of restriction fragment length polymorphism (RFLP) analysis of PCV2 open reading frame 2 for easy discrimination between the two major groups. Genotyping of PCV2 isolates from 10 Japanese commercial pig farms was performed, and the analysis revealed that both PCV2 groups and at least five RFLP types of PCV2 are prevalent in Japan. On two farms, the genotypes of the PCV2 isolates in the spring of 2007 were different from those in the autumn of 2006. One genotype may have become dominant within only six months on these farms.

Genomic analysis of PCV2 isolates from Danish archives and a current PMWS case–control study supports a shift in genotypes with time

Porcine circovirus type 2 (PCV2) is the primary cause of Postweaning Multisystemic Wasting Syndrome (PMWS) in pigs. PCV2, however, is found in both PMWS-affected herds and non-affected herds. The objective of this study was to clarify if PCV2 genome nucleotide sequences isolated from pigs from PMWS-affected herds and non-affected herds cluster phylogenetically in two separate groups. All isolates (45) belonged to PCV2 group 1 and shared a nucleotide sequence identity of 99.4–100% indicating a very homogeneous PCV2 population in Denmark. Phylogenetic analysis of the PCV2 isolates revealed no distinctive clustering of case- and control-herds suggesting that there is no link between PCV2 sequences and herd disease status. The appearance of only PCV2 group 1 isolates in this study (isolates from 2003/2004) led us to determine if PCV2 nucleotide sequences had changed in Denmark over time. Interestingly, all PCV2 isolates from before the first outbreak of PMWS (2001) belonged either to a new PCV2 group identified for the first time in this study and named group 3 (isolates from 1980, 1987 and 1990) or PCV2 group 2 (isolates from 1993 and 1996). The shift from PCV2 group 2 to 1 was confirmed on a more global scale by placing all full genome PCV2 sequences submitted to GenBank from 1997 to 2006 in either of the groups by phylogenetic analysis. The analysis showed that the shift happened in 2003 or even earlier. This may indicate that PCV2 group 1 is a more adapted form of PCV2 and possibly could be more pathogenic.