Evidence of porcine circovirus type 2 and co-infection with ungulate protoparvovirus 1 (porcine parvovirus) in mummies and stillborn piglets in subclinically infected farm.

Porcine circovirus type 2 (PCV2) and protoparvovirus 1 (PPV) were detected as single infection (6/131) and (11/131) respectively, or co-infection (6/131) in fetuses and stillborn piglets from normal deliveries in a farm without reproductive problems. Twenty in twenty-three positive samples were over 70 days of gestation, which is when the fetus becomes immunocompetent, and the presence of a NADL-2 PPV strain suggests fetal immune system impairment. Phylogenetic analysis of sequences obtained showed that 8/9 sequences are related to cluster 13 and the remaining is grouped into cluster 11 sequences. An increase in variability in ORF2 sequences in Argentina was observed. It is not clear whether the detection of fetuses positive to PPV and PCV2 is of epidemiological importance in a subclinically affected farm. However, the results of this study showed that currently used vaccines and vaccine protocols do not fully protect against PPV or PCV2 fetus infection.

The effect of Panax notoginseng saponins on oxidative stress induced by PCV2 infection in immune cells: in vitro and in vivo studies.

BACKGROUND: Panax notoginseng saponins (PNS) are bioactive substances extracted from P. notoginseng that are widely used to treat cardiovascular and cerebrovascular diseases and interstitial diseases. PNS have the functions of scavenging free radicals, anti-inflammation, improving blood supply for tissue and so on. OBJECTIVES: The aim of this study was to investigate the effects of PNS on the oxidative stress of immune cells induced by porcine circovirus 2 (PCV2) infection in vitro and in vivo. METHODS: Using an oxidative stress model of PCV2 infection in a porcine lung cell line (3D4/2 cells) and mice, the levels of nitric oxide (NO), reactive oxygen species (ROS), total glutathione (T-GSH), reduced glutathione (GSH), and oxidized glutathione (GSSG) and the activities of xanthine oxidase (XOD), myeloperoxidase (MPO) and inducible nitric oxide synthetase (iNOS) were determined to evaluate the regulatory effects of PNS on oxidative stress. RESULTS: PNS treatment significantly reduced the levels of NO and ROS, the content of GSSG and the activities of XOD, MPO, and iNOS (p < 0.05), while significantly increasing GSH and the ratio of GSH/GSSG in infected 3D4/2 cells (p < 0.05).Similarly, in the in vivo study, PNS treatment significantly decreased the level of ROS in spleen lymphocytes of infected mice (p < 0.05), increased the levels of GSH and T-GSH (p < 0.05), significantly decreased the GSSG level (p < 0.05), and decreased the activities of XOD, MPO, and iNOS. CONCLUSIONS: PNS could regulate the oxidative stress of immune cells induced by PCV2 infection in vitro and in vivo.

Haemato-biochemical alterations and oxidative stress associated with naturally occurring porcine circovirus2 infection in pigs.

Porcine circovirus2 (PCV2) infection in pigs is one of the major causes of economic loss to the farmers in terms of low production, slow growth and increase post-weaning mortality rate. The effect of PCV2 infection on haemogram, serum biochemical profile and oxidant/anti-oxidant status is not well established in pigs. In the present study, haemogram, serum biochemical profile and oxidant/anti-oxidant status were assessed in pigs confirmed positive for PCV2 infections as evidenced by commercially available enzyme-linked immunosorbent assay kit (n = 151) and polymerase chain reaction (PCR) (n = 42) among a total of 306 number of pigs included in the study. Non-infected healthy pigs (n = 6) served as healthy control. The total erythrocyte count (TEC), haemoglobin (Hb), packed cell volume (PCV), total leukocyte count (TLC), differential leukocyte count (DLC) and thrombocyte count were measured. The levels of total protein, albumin, globulin, total bilirubin, direct bilirubin, blood urea nitrogen (BUN), creatinine and glucose and enzymes viz. alanine transaminase (ALT), aspartate transaminase (AST), gamma-glutamyl transferase (GGT) and alkaline phosphatase (ALP) were measured. Oxidative stress indicators such as plasma malondialdehyde (MDA) and total anti-oxidant activity (TAOA) were measured using commercially available kits. The mean values of TLC, lymphocytes and thrombocyte count were significantly (P < 0.05) low in PCV2-infected pigs. The levels of globulin, AST, GGT, BUN and creatinine were significantly increased (P < 0.05) whereas levels of albumin and glucose significantly (P < 0.05) decreased in PCV2-infected pigs. The significant increase (P < 0.05) in MDA level and significant decrease (P < 0.05) in TAOA level were noticed in PCV2-infected animals as compared with healthy control. The present study supports immunosuppression, possible multiple organ damage and oxidative stress associated with naturally occurring PCV2 infection in pigs. Timely vaccination and managemental practices can reduce PCV2 infection in farms. In spite of many research studies, there is still paucity of detailed systemic study on haemato-biochemical alteration and oxidative stress associated with PCV2 infection.

Molecular detection and sequence analysis of porcine circovirus type 3 in sow sera from farms with prolonged histories of reproductive problems in Hunan, China.

A newly emerging porcine circovirus, designated PCV3, has been reported in various countries (USA, Poland, South Korea and China) since 2017. Its presence may be associated with porcine dermatitis and nephropathy syndrome (PDNS), reproductive failure, and multisystem inflammation. In this study, we report identification of PCV3 in cases of reproductive failure in various regions in Hunan, China. From January 2015 to December 2016, sera were collected from 190 sows from seven farms with reproductive problems. Specifically, 85 samples were from sows with a history of reproductive failure, whereas the remaining 105 were from healthy sows. The PCV3-positive rate was significantly higher in sows with reproductive failure (45.9%) than in healthy sows (21.9%), based on quantitative PCR (qPCR) assays. Although phylogenetic analysis based on the cap gene suggested that these PCV3 isolates belonged to the clade PCV3a, amino acid sequence variations in the Cap protein still occurred among these isolates, and these might have contributed to antigenic alterations of the Cap protein, based on the Jameson-Wolf antigenic index. Finally, we concluded that PCV3 was circulating in sows in Hunan province, China. However, the association of PCV3 with reproductive failure in sows and its potential for vertical transmission need to be studied further.

Porcine Circovirus 2 Deploys PERK Pathway and GRP78 for Its Enhanced Replication in PK-15 Cells.

Porcine circovirus type 2 (PCV2) infection induces autophagy and apoptosis. These cellular responses could be connected with endoplasmic reticulum (ER) stress. It remains unknown if PCV2 induces ER stress and if autophagy or apoptosis is primary to PCV2 infection or secondary responses following ER stress. Here, we demonstrate that PCV2 triggered unfolded protein response (UPR) in PK-15 cells by activating the PERK/eIF2α pathway without concomitant activation of IRE1 or ATF6. Since ATF4 and CHOP were induced later than PERK/eIF2α, it is clear that persistent PCV2 infection could lead to selective activation of PERK via the PERK-eIF2α-ATF4-CHOP axis. Therefore, PERK activation could be part of the pro-apoptotic signaling via induced expression of CHOP by PCV2. Since PERK inhibition by GSK2606414 or RNA silencing or suppression of eIF2α dephosphorylation by salubrinal limited viral replication, we suppose that PCV2 deploys UPR to enhance its replication. Over-expression of GRP78 or treatment with tauroursodeoxycholic acid could enhance viral capsid expression and/or viral titers, indicating that these chaperones, endogenous or exogenous, could help correct folding of viral proteins. Our findings provide the first evidence that ER stress plays a role in the pathogenesis of PCV2 infection probably as part of autophagic and apoptotic responses.

Characterization of specific antigenic epitopes and the nuclear export signal of the Porcine circovirus 2 ORF3 protein.

Porcine circovirus 2 (PCV2) is the etiological agent of postweaning multisystemic wasting syndrome. PCV2 ORF3 protein is a nonstructural protein known to induce apoptosis, but little is known about the biological function of ORF3 protein. Therefore, we undertook this study to map ORF3 protein epitopes recognized by a panel of monoclonal antibodies (mAbs) and to characterize putative nuclear localization (NLS) and nuclear export (NES) sequences in ORF3. The linear epitopes targeted by two previously published mAbs 3B1 and 1H3 and a novel mouse mAb 3C3 were defined using overlapping pools of peptides. Here, we find that ORF3 in PCV2 infected cells contains a conformational epitope targeted by the antibody 3C3, which is distinct from linear epitopes recognized by the antibodies 3B1 and 1H3 in recombinant ORF3 protein. These results suggest that the linear epitope recognized by 3B1 and 1H3 is masked in PCV2 infected cells, and that the conformational epitope is unique to PCV2 infection. Furthermore, we find that ORF3 protein expressed in cytoplasm in early stages of PCV2 infection and then accumulated in nucleus over time. Moreover, we localize a NES at the N-terminus (residues 1-35aa) of ORF3 which plays critical role in nuclear export activity. These findings provide a novel insight that deepens our understanding of the biological function of PCV2 ORF3.

Evidence of apoptosis induced by viral protein 2 of chicken anaemia virus.

Although viral protein 3 (VP3) of chicken anaemia virus (CAV) has been well recognised as an inducer of apoptosis, viral protein 2 (VP2) of the virus has only been speculated to have apoptotic activity. This has not been verified because the open reading frame (ORF) encoding VP2 completely encompasses that encoding VP3, and thus the possibility of expression of VP3 cannot be excluded. The aim of this study was to elucidate the potential role of VP2 as an inducer of apoptosis. Site-directed mutagenesis was used to generate a point mutation that knocked out VP3 by early termination of its translation with a stop codon without imposing any change in the amino acid sequence of VP2. The mutated sequence was inserted into the pCAT plasmid preceded by a favorable Kozak's consensus sequence to create pCAT-VP2(+)VP3(-). The absence of VP3 expression in MSB1 cells transfected with this plasmid was confirmed using Western blotting, and DNA strand breaks and nuclear morphological changes were assessed to detect apoptosis. There was an increased level of apoptotic death in cells transfected with pCAT-VP2(+)VP3(-) compared to those transfected with the vector alone. This provides evidence that CAV VP2 can induce apoptosis.

Exposure to environmental stressors result in increased viral load and further reduction of production parameters in pigs experimentally infected with PCV2b.

Porcine circovirus type 2 (PCV2) has been identified as the essential, but not sole, underlying infectious component for PCV-associated diseases (PCVAD). Several co-factors have been suggested to convert an infection with PCV2 into the clinical signs of PCVAD, including co-infection with a secondary pathogen and the genetic background of the pig. In the present study, we investigated the role of environmental stressors in the form of changes in environmental temperature and increased stocking-density on viral load in serum and tissue, average daily weight gain (ADG) and food conversion rate (FCR) of pigs experimentally infected with a defined PCV2b strain over an eight week period. These stressors were identified recently as risk factors leading to the occurrence of severe PCVAD on a farm level. In the current study, PCV2-free pigs were housed in separate, environmentally controlled rooms, and the experiment was performed in a 2×2 factorial design. In general, PCV2b infection reduced ADG and increased FCR, and these were further impacted on by the environmental stressors. Furthermore, all stressors led to an increased viral load in serum and tissue as assessed by qPCR, although levels did not reach statistical significance. Our data suggest that there is no need for an additional pathogen to develop PCVAD in conventional status pigs, and growth retardation and clinical signs can be induced in PCV2 infected pigs that are exposed to environmental stressors alone.

Porcine CD74 is involved in the inflammatory response activated by nuclear factor kappa B during porcine circovirus type 2 (PCV-2) infection.

Human CD74 induces a signalling cascade that results in the activation of nuclear factor kappa B (NF-κB); however, porcine CD74 has not been widely studied. In this study, we show that porcine CD74 is mainly expressed in cells of the macrophage lineage and can be induced by lipopolysaccharide (LPS), polyinosinic acid-polycytidylic acid [Poly(I:C)], and infection with porcine circovirus type 2 (PCV2) in vitro. In addition, we confirmed that porcine CD74 can activate NF-κB by promoting IκBα degradation and nuclear translocation of p65. Furthermore, the transcription of NF-κB-regulated genes [Interleukin-6 (IL-6), Interleukin-8 (IL-8), and COX-2] was upregulated in response to the overexpression of porcine CD74. In general, porcine CD74 significantly enhanced the inflammatory response by regulating the NF-κB signalling pathway during PCV2 infection, which suggests that porcine CD74 may be implicated in the pathogenesis of PCV2 infection.

Recognition of the different structural forms of the capsid protein determines the outcome following infection with porcine circovirus type 2.

Porcine circovirus type 2 (PCV2) capsid protein (CP) is the only protein necessary for the formation of the virion capsid, and recombinant CP spontaneously forms virus-like particles (VLPs). Located within a single CP subunit is an immunodominant epitope consisting of residues 169 to 180 [CP(169-180)], which is exposed on the surface of the subunit, but, in the structural context of the VLP, the epitope is buried and inaccessible to antibody. High levels of anti-CP(169-180) activity are associated with porcine circovirus-associated disease (PCVAD). The purpose of this study was to investigate the role of the immune response to monomer CP in the development of PCVAD. The approach was to immunize pigs with CP monomer, followed by challenge with PCV2 and porcine reproductive and respiratory syndrome virus (PRRSV). To maintain the CP immunogen as a stable monomer, CP(43-233) was fused to ubiquitin (Ub-CP). Size exclusion chromatography showed that Ub-CP was present as a single 33-kDa protein. Pigs immunized with Ub-CP developed a strong antibody response to PCV2, including antibodies against CP(169-180). However, only low levels of virus neutralizing activity were detected, and viremia levels were similar to those of nonimmunized pigs. As a positive control, immunization with baculovirus-expressed CP (Bac-CP) resulted in high levels of virus neutralizing activity, small amounts of anti-CP(169-180) activity, and the absence of viremia in pigs following virus challenge. The data support the role of CP(169-180) as an immunological decoy and illustrate the importance of the structural form of the CP immunogen in determining the outcome following infection.

Porcine circovirus 2 (PCV2) induces a procoagulant state in naturally infected swine and in cultured endothelial cells.

Porcine circovirus 2 (PCV2) is the primary causative agent of porcine circovirus disease (PCVD). PCVD is an emerging disease that has been reported worldwide, associated with wasting, lymphoid depletion, enteritis, pneumonia, vasculitis, ischemic lesions, and necrotizing dermatitis. Although PCVD causes considerable economic losses, the pathogenesis of PCV2 has not been fully understood. The aim of the present work was to study the participation of hemostatic system and of vascular endothelium in PCV2 infection, as well as their possible role in PCVD pathogenesis. Our results showed that naturally PCV2-infected swine displayed a prothrombotic state in vivo, since a diminished coagulation time (recalcification time, activated partial thromboplastin time and prothrombin time), a higher platelet aggregation ability (despite a diminished platelet blood count), and an increased thrombin plasma activity (associated with a reduced fibrinogen level) were observed. The PCV2-infected animals showed vasculitis and positive staining for PCV2 antigen in capillary vessels. Furthermore, PCV2-infected endothelial cells displayed an activated phenotype, characterized by an increase in cell surface procoagulant activity. Moreover, the PCV2-infected endothelial cells pre-treated with exogenous thrombin displayed an increased viral load. This work reports, for the first time, the role of the hemostatic system and of endothelium in the pathogenesis and infectivity of PCV2. The study reinforces the importance of the phenomena which occur during PCV2 infection, and affords a better knowledge of the mechanisms behind the pathophysiology of PCVD.

Reproductive failure experimentally induced in sows via artificial insemination with semen spiked with porcine circovirus type 2.

Porcine circovirus type 2 (PCV2) is associated with reproductive failure in female pigs. However, the association of PCV2-positive semen in the pathogenesis has not been elucidated. The objectives of this study were to determine whether semen spiked with PCV2 causes infection in PCV2-naïve, mature female pigs and whether delivery of PCV2 via artificial insemination causes reproductive failure or fetal infection. Nine sows were randomly allocated into 3 groups of 3 sows each and artificially inseminated with PCV2 DNA-negative semen (group 1), PCV2 DNA-negative semen spiked with PCV2a (group 2), or PCV2b (group 3). All sows in groups 2 and 3 developed PCV2 viremia 7 to 14 days after insemination. None of the group 2 sows became pregnant, whereas all group 3 sows (3/3) farrowed at the expected date. At parturition, presuckle serum samples were collected, and live-born piglets, stillborn fetuses, and mummified fetuses were necropsied. All live-born piglets (n = 8) in group 3 were PCV2 viremic at birth. Stillborn fetuses (n = 2) had gross lesions of congestive heart failure. Mummified fetuses (n = 25) varied in crown-rump length from 7 to 27 cm, indicating fetal death between 42 and 105 days of gestation. PCV2 antigen was detected in the myocardium by immunohistochemistry of 7/8 (88%) live-born piglets, 2/2 (100%) of the stillborn fetuses, and 25/25 (100%) of the mummified fetuses. In addition, 4/25 mummified fetuses had PCV2 antigen associated with smooth muscle cells and fibroblasts. The results of this study indicate that intrauterine administration of PCV2 causes reproductive failure in naïve sows.

Porcine circovirus 2 infection of epithelial cells is clathrin-, caveolae- and dynamin-independent, actin and Rho-GTPase-mediated, and enhanced by cholesterol depletion.

Epithelial cells are the major in vivo target cells for porcine circovirus type 2 (PCV2). Although these cells are used for most studies of PCV2 gene expression and, little is known on PCV2 entry, attachment and internalization, in epithelial cells. PCV2 attachment to epithelial cells occurred rapidly and in a time-dependent manner. In contrast to attachment, internalization was slow. Immunofluorescent stainings revealed that during internalization, PCV2 co-localized with clathrin, but not caveolin. Blocking clathrin-mediated endocytosis increased instead of decreased the number of PCV2-infected cells by threefold, suggesting that it does not represent the main internalization pathway leading to a full replication. Further analysis with different inhibitors revealed that also macropinocytosis, dynamin-dependent internalization and membrane cholesterol play no role in PCV2 entry that leads to infection. Inhibition of small GTPases with Clostridium difficile toxin B reduced the number of PCV2-infected PK-15, SK and STs to 63+/-25%, 47+/-21% and 14+/-6%, respectively. Finally, inhibiting actin polymerization also blocked PCV2 infection, showing the need for actin during PCV2 infection. Together, these data indicate that a dynamin- and cholesterol-independent, but actin- and small GTPase-dependent pathway, allows PCV2 internalization in epithelial cells that leads to infection and that clathrin-mediated PCV2 internalization in epithelial cells is not followed by a full replication.

Differences in virulence among porcine circovirus type 2 isolates are unrelated to cluster type 2a or 2b and prior infection provides heterologous protection.

Porcine circovirus type 2 (PCV2) is divided into two genetic clusters designated PCV2a and PCV2b. The objectives of this study were to determine whether isolates from different clusters vary in virulence and to determine whether infection with PCV2a isolates induces protective immunity against subsequent infection with a recent PCV2b isolate. One-hundred and thirteen conventional specific-pathogen-free (SPF) pigs were assigned randomly to treatment groups and rooms: pigs inoculated with PCV2a cluster isolates (ISU-40895 or ISU-4838), pigs inoculated with PCV2b cluster isolates (NC-16845 or Can-17639) and uninoculated pigs. Necropsies were performed at 16 or 51 days post-inoculation (p.i.). There were no significant differences in PCV2-associated lymphoid lesions between PCV2a and PCV2b clusters; however, within the same cluster, significant differences were found between isolates: ISU-4838- and Can-17639-inoculated pigs had significantly (P<0.05) less severe lesions compared with ISU-40895- and NC-16845-inoculated pigs. To evaluate cross-protection, six pigs within each group were challenged at 35 days p.i. with an isolate from the heterologous cluster and were necropsied 51 days p.i. The severity of PCV2-associated lesions was reduced in pigs with prior exposure to an isolate from the heterologous cluster in comparison with singly inoculated pigs. Results indicate that the virulence of PCV2a and PCV2b isolates is not different in the conventional SPF pig model; however, the virulence of isolates within the same cluster differs. Increased virulence as reported to be associated with PCV2b isolates in the field was not observed under the conditions of this study. Moreover, cross-protection between PCV2a and PCV2b exists.

Reduction of PMWS-associated clinical signs and co-infections by vaccination against PCV2.

The effects of a single-dose recombinant Porcine circovirus type 2 (PCV2) open reading frame 2 (ORF2) subunit vaccine were studied in a post-weaning multisystemic wasting syndrome (PMWS)-affected pig herd. A total of 1519 3-week-old piglets were allocated randomly into two treatment groups and either vaccinated against PCV2 or treated with a placebo. Study animals were followed from the time of vaccination until the end of finishing. Onset of PCV2 viraemia and clinical signs of PMWS (wasting, cough, dyspnoea, pallor and lethargy) were observed when animals were approximately 9-10 weeks old. Compared to placebo-treated animals, vaccinated animals had a significantly reduced PCV2 viral load and duration of viraemia (p < 0.0001). This reduction in viraemia was not affected by the level of maternal anti-PCV2 antibodies present at the time of vaccination. During the period of viraemia (10-26 weeks of age) vaccinated animals exhibited a 53% reduction in mortality rate (p = 0.0010), a 4.84 kg higher body weight gain (p < 0.0001) and a significant reduction in clinical signs (p < or = 0.0004). Furthermore, lung samples of vaccinated animals had a considerably reduced number of co-infections with PRRSV and Mycoplasma hyorhinis than lung samples of placebo-treated animals. These data indicate that vaccination against PCV2 alone protects pigs from clinical signs and co-infections associated with PMWS.

Cytokine and C-reactive protein profiles induced by porcine circovirus type 2 experimental infection in 3-week-old piglets.

The purpose of this study was to determine serum profiles of cytokines at a protein level and Creactive protein (CRP) during the development of postweaning multisystemic wasting syndrome (PMWS) in experimentally inoculated pigs. Levels of serum IFN-alpha, IL-6, IL-10, and CRP were examined for a 35-day period in 10 piglets experimentally infected with PCV2 at 3 weeks of age. Four of the infected piglets developed severe PMWS at 14 to 21 days post-infection (d.p.i.) and died prior to termination of the experiment. The remaining six PCV2-infected piglets experienced transient fever, but did not display overt clinical signs of PMWS and were considered as subclinically infected. A bioassay was used to detect IL-6 and ELISAs were used to detect IFN-alpha, IL-10, and CRP. There were no significant differences in cytokine or CRP expression from 0 to 7 d.p.i. between the PMWS-affected and the subclinically infected piglets. Levels of IL-10 and CRP were elevated from 10 and 14 d.p.i. respectively in the PMWS-affected piglets compared to the subclinically infected piglets. There were no significant differences in IFN-alpha and IL-6 expression between the PMWS-affected piglets and the subclinically infected piglets. The present study shows that elevated levels of serum CRP and IL-10 were associated with PCV2-infected piglets that subsequently developed severe PMWS. This may help to provide further insight into the immunoaetiogenesis of this syndrome.

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.

Porcine circovirus type 2 (PCV2) is the primary causative agent of postweaning multisystemic wasting syndrome (PMWS) in pigs. To identify potential genetic determinants for virulence and replication, we serially passaged a PCV2 isolate 120 times in PK-15 cells. The viruses harvested at virus passages 1 (VP1) and 120 (VP120) were biologically, genetically, and experimentally characterized. The PCV2 VP120 virus replicated in PK-15 cells to a titer similar to that of the PK-15 cell line-derived nonpathogenic PCV1 but replicated more efficiently than PCV2 VP1 with a difference of about 1 log unit in the titers. The complete genomic sequences of viruses at passages 0, 30, 60, 90, and 120 were determined. After 120 passages, only two nucleotide mutations were identified in the entire genome, and both were located in the capsid gene: the mutations were located at nucleotide positions 328 (C328G) and 573 (A573C). The C328G mutation, in which a proline at position 110 of the capsid protein changed to an alanine (P110A), occurred at passage 30 and remained in the subsequent passages. The second mutation, A573C, resulting in a change from an arginine to a serine at position 191 (R191S), appeared at passage 120. To experimentally characterize the VP120 virus, 31 specific-pathogen-free pigs were randomly divided into three groups. Ten pigs in group 1 received phosphate-buffered saline as negative controls. Each pig in group 2 (11 pigs) was inoculated intramuscularly and intranasally with 10(4.9) 50% tissue culture infective doses (TCID(50)) of PCV2 VP120. Each pig in group 3 (10 pigs) was similarly inoculated with 10(4.9) TCID(50) of PCV2 VP1. Viremia was detected in 9 of 10 pigs in the PCV2 VP1 group with a mean duration of 3 weeks, but in only 4 of 11 pigs in the PCV2 VP120 group with a mean duration of 1.6 weeks. The PCV2 genomic copy numbers in serum in the PCV2 VP1 group were significantly higher than those in the PCV2 VP120 group (P < 0.0001). Gross and histopathologic lesions in pigs inoculated with PCV2 VP1 were more severe than those inoculated with PCV2 VP120 at both day 21 and 42 necropsies (P = 0.0032 and P = 0.0274, respectively). Taken together, the results from this study indicated that the P110A and R191S mutations in the capsid of PCV2 enhanced the growth ability of PCV2 in vitro and attenuated the virus in vivo. This finding has important implications for PCV2 vaccine development.

Pathogenesis of chicken anemia virus: comparison of the oral and the intramuscular routes of infection.

The events during the pathogenesis of chicken anemia virus (CAV) infection following intramuscular (IM) and oral inoculation were further elucidated and compared by sequential clinical, pathologic, and morphometric histopathologic evaluations, and by sequential determination of CAV genome concentrations in different organs. Specific-pathogen-free chickens were inoculated by IM or oral routes with the same dose (2 x 10(6) mean tissue culture infective dose [TCID50]) of CAV isolate 03-4876 at 1 day of age. Weights and hematocrits were obtained at 7, 10, 14, 18, 21, 25, and 28 days postinoculation (DPI). Seven birds from each group were necropsied at 7, 10, 14, and 28 DPI, and samples of thymus, Harderian gland, and cecal tonsils (CT) were obtained for histopathologic examination and CAV genome quantification by real-time polymerase chain reaction. Peak CAV genome concentrations were detected in the thymus at 10 and 14 DPI in the IM and orally infected chickens, respectively. High CAV DNA concentrations were maintained throughout the experimental period until 28 DPI, despite specific seroconversion occurring by 14 DPI in the IM-inoculated chickens. CAV was isolated from both orally and IM-infected chickens 28 DPI. Peak CAV genomes in the thymuses of IM and orally infected chickens coincided with peak lymphocyte depletion in these organs. Lymphocyte repopulation of the thymus occurred by 28 DPI in spite of the presence of the virus in the organs of both infected chicken groups. CAV genomes were detected in the CT, but histopathologic changes were not observed. Compared with the IM route of infection, orally infected chickens did not show apparent signs of illness. Clinical parameters, including reduction of weight gains and hematocrits, and gross and histopathologic changes were delayed and less severe in the orally inoculated chickens. This was concurrent with a delay in accumulation of CAV genomes in the thymus of these chickens.

Reproduction of postweaning multisystemic wasting syndrome in pigs experimentally inoculated with a Swedish porcine circovirus 2 isolate.

In recent years, porcine circovirus type 2 (PCV2)-associated postweaning multisystemic wasting syndrome (PMWS) has been reported worldwide. However, to date, PMWS has not been reported in Sweden despite the demonstration of serum antibodies to a PCV2-like virus in Swedish pigs. This communication reports the experimental reproduction of clinical PMWS after inoculation of colostrum-deprived (CD) pigs, derived from a Northern Ireland herd, with an isolate of PCV2 virus recovered from a clinically normal Swedish pig that was necropsied in 1993. The clinical disease and histological lesions observed in CD pigs inoculated with this virus were indistinguishable from those observed in previous studies on CD pigs inoculated with a PCV2 virus isolate recovered from pigs with PMWS. These results highlight the disease potential of PCV2 isolated from regions apparently free of PMWS and suggest that the status of the host and its environment is an important factor in the development of clinical PMWS.

Protection of swine against post-weaning multisystemic wasting syndrome (PMWS) by porcine circovirus type 2 (PCV2) proteins.

Porcine circovirus type 2 (PCV2) is known to be associated with post-weaning multisystemic wasting syndrome (PMWS), a recently described disease of young pigs. Since no PCV2 vaccine was available so far, we have developed a specific PCV2 vaccine candidate. The Orf1-encoded replication protein and Orf2-encoded capsid protein of PCV2 were expressed and detected in either mammalian or insect expression systems. In a first trial, Orf2 protein was found to be a major immunogen, inducing protection in a prime-boost protocol; the piglets received a first injection with plasmids directing Orf2 protein and granulocyte-macrophage colony-stimulating factor (GM-CSF) expression, followed by a second injection, a fortnight later, associated with baculovirus-expressed Orf2 protein. As evaluated by growth parameters, clinical signs (fever), seroconversion, the pigs were protected against a PCV2 challenge after vaccination. In a second trial, protection induced by a subunit vaccine was even better than the one induced by DNA vaccine, since PCV2 replication was completely inhibited.