Porcine circovirus type 2 morphogenesis in a clone derived from the l35 lymphoblastoid cell line.

Porcine circovirus type 2 (PCV2) is the essential infectious agent of post-weaning multisystemic wasting syndrome (PMWS), one of the most important diseases of swine. Although several studies have described different biological properties of the virus, some aspects of its replication cycle, including ultrastructural alterations, remain unknown. The aim of the present study was to describe for the first time a complete morphogenesis study of PCV2 in a clone of the lymphoblastoid L35 cell line at the ultrastructural level using electron microscopy techniques. Cells were infected with PCV2 at a multiplicity of infection of 10 and examined at 0, 6, 12, 24, 48, 60 and 72h post-infection. PCV2 was internalized by endocytosis, after which the virus aggregated in intracytoplasmic inclusion bodies (ICIs). Subsequently, PCV2 was closely associated with mitochondria, completing a first cytoplasmic phase. The virus entered the nucleus for replication and virus assembly and encapsidation occurred with the participation of the nuclear membrane. Immature virions left the nucleus and formed ICIs in a second cytoplasmic phase. The results suggest that at the end of the replication cycle (between 24 and 48h), PCV2 was released either by budding of mature virion clusters or by lysis of apoptotic or dead cells. In conclusion, the L35-derived clone represents a suitable in-vitro model for PCV2 morphogenesis studies and characterization of the PCV2 replication cycle.

Immune responses and vaccine-induced immunity against Porcine circovirus type 2.

Porcine circovirus type 2 (PCV2) is essential but not sufficient for postweaning multisystemic wasting syndrome (PMWS) occurrence in pigs. The outcome of PCV2 infection depends on the specific immune responses that are developing during the infection. Diseased pigs are immunosupressed and unable to mount effective immune responses to clear the virus from circulation. In the final stage, PMWS-affected pigs suffer from extensive lymphoid lesions and altered cytokine expression patterns in peripheral blood mononuclear cells (PBMCs) and lymphoid organs. PCV2 infection can also be asymptomatic, demonstrating that not every infection will guarantee the occurrence of severe immunopathological disturbances. Asymptomatic animals have higher virus specific and neutralising antibody titres than PMWS-affected animals. Recent results have pointed out that the mechanisms by which PCV2 can affect the immune responses involve the induction of IL-10, virus accumulation into and modulation of plasmacytoid dendritic cells and the role of viral DNA in regulation of immune cell functions. Fourteen years after the first description of PMWS in Canada, efficient commercial vaccines against PCV2 are available. The vaccine success is based on activated humoral and cellular immune responses against PCV2. This review focuses on the recent research on immunological aspects during PCV2 infections and summarizes what is currently known about the vaccine-induced immunity.

Porcine circovirus 2 replication in colostrum-deprived piglets following experimental infection and immune stimulation using a modified live vaccine against porcine respiratory and reproductive syndrome virus.

Porcine circovirus type 2 (PCV2) infection is now recognized as the major factor in the development of post-weaning multisystemic wasting syndrome (PMWS). Although Koch's postulates have been fulfilled for PCV2 and PMWS, the severe clinical expression of the disease observed in field cases has been difficult to reproduce experimentally. Some studies have demonstrated that immune stimulation associated with the use of some commercially available swine vaccines may trigger progression of PCV2 infection to disease and lesions characteristic of PMWS. Here we describe the effects on PCV2 infection in an experimental model following the use of a commercially available modified live vaccine to porcine respiratory and reproductive syndrome virus (PRRSV). Although none of the piglets infected with PCV2 developed clinical PMWS, the severity of microscopical lesions and the PCV2 antigen load associated with these lesions were higher in the PRRSV-vaccinated piglets compared with those detected in the PCV2 only infected animals.

Postweaning multisystemic wasting syndrome (PMWS) in Sweden from an exotic to an endemic disease.

Postweaning multisystemic wasting syndrome (PMWS) is causally associated with porcine circovirus type 2 (PCV2) infection of pigs. PCV2 was first demonstrated in Swedish pigs in 1993, although the virus was almost certainly present in pigs in the country before that. Despite this, no signs of PMWS were observed in pigs of Sweden until the first outbreak was reported in 2003. The accumulated number of PMWS-affected herds have increased via 16 (2004) and 41 (2005) to 123 in December 2006. Of these herds, 30 (25%) have now been declared free from PMWS. However, a number of other herds have had individual pigs that have fulfilled the demands for PMWS at necropsy and 52 of these herds have been declared negative on herd basis after treatment for intestinal or respiratory diseases, and/or by correcting shortcomings in management of the herd including feed. Thus, individual cases of the disease have been observed in around 200 herds by the end of 2006 and PMWS is now regarded as an endemic disease in Sweden. The pig population of Sweden is geographically isolated, the density of pigs and the pathogen load in the country is low and the use of growth promoters (low dose antibiotics in feed) was prohibited in 1986. Additionally, the trade of animals in Sweden is organised in a restricted way. Because of these factors it is possible to conduct meaningful real-time studies on the transformation of PMWS in Sweden from being an exotic to an endemic disease in a three year time scale. Initially the PMWS cases were concentrated in the southern part of Sweden, but have gradually spread north. The PMWS-positive herds have, in general, had an effective production, but some management errors have constantly been observed in affected herds. Physical links between affected herds are often missing, and the data generated to date on the PMWS outbreaks in Sweden do not suggest an introduction of a new contagious microbe into the country that is responsible for the PMWS outbreaks, nor does PMWS appear to be spread via semen. In Sweden, intensity in rearing, disease preventing measures and immaturity of the piglets appear to be important as predisposing factors to PMWS and, as such, are discussed in this article.

The regulation and inhibition of 17beta-hydroxysteroid dehydrogenase in breast cancer.

17Beta-hydroxysteroid dehydrogenase Type 1 (17beta-HSD1) has a pivotal role in regulating the synthesis of oestradiol (E2) within breast tumours. In whole body studies in postmenopausal women with breast cancer the conversion of oestrone (E1) to E2 (4.4+/-1.1%) was much lower than the inactivation of E2 to E1 (17.3+/-5.0%). In contrast, an examination of in vivo oestrogen metabolism within breast tumours revealed that whereas little metabolism of E2 occurred, E1 was converted to E2 to a much greater extent in malignant (48+/-14%) than in normal (19+/-6%) breast tissue. Findings from these studies originally suggested that oestrogen metabolism within breast tumours may differ from the mainly oxidative direction found in most other body tissues and that the activity of 17beta-HSD1 might be regulated by tumour-derived factors. Several growth factors (e.g. IGF-I, IGF-II) and cytokines (e.g. IL-6, TNFalpha) have now been identified which can markedly stimulate the activity of 17beta-HSD1 and such a mechanism may account for the high concentrations of E2 found in most breast tumours. Cells of the immune system, which can infiltrate breast tumours, are thought to be a major source of the growth factors and cytokines which can modulate 17beta-HSD1 activity. Given the central role that 17beta-HSD1 has in regulating breast tumour E2 concentrations the development of potent inhibitors of this enzyme has recently attracted considerable attention. Our initial studies in this area explored the use of derivatives of E1 as inhibitors, with 2-ethyl- and 2-methoxy E1 being found to inhibit 17beta-HSD1 activity in T-47D breast cancer cells by 96+/-2 and 91+/-1% respectively at 10 microM, but with a lack of specificity. Using the E1 scaffold a number of potent, selective 17beta-HSD1 inhibitors have now been identified including E1- and 2-ethyl-E1 containing a side chain with a m-pyridylmethylamidomethyl functionality extending from the 16beta position of the steroid nucleus. At 10 microM these compounds both inhibited 17beta-HSD1 activity by >90%, however some inhibition of 17beta-HSD2 activity was exhibited by the E1 derivative (25%) but not the 2-ethyl analogue. It is now apparent that 17beta-HSD1 activity contributes to the high E2 concentrations found in most breast tumours. The identification of potent, selective novel 17beta-HSD1 inhibitors will allow their efficacy to be tested in in vitro and in vivo studies.

Experimental infection of 3-week-old conventional colostrum-fed pigs with porcine circovirus type 2 and porcine parvovirus.

This report describes an experimental infection with porcine circovirus type 2 (PCV2) in combination with porcine parvovirus (PPV) in 3-week-old conventional colostrum-fed pigs with maternal antibodies to both viruses. Two groups of four pigs each were inoculated with PCV2 and PPV. One of the groups received also a commercial inactivated vaccine against porcine pleuropneumonia to evaluate possible effects of the stimulation of the immune system of pigs on the infection. Another group of four pigs was kept as uninfected control. Clinical signs, rectal temperatures and body weights were recorded. Serum antibody titers to PCV2 and PPV were determined at weekly intervals. Pigs were killed 42 days after inoculation and tissue samples were examined for the presence of gross and microscopic lesions. Tissues were also analyzed for the presence of PCV2 and PPV DNA by PCR, and for the presence of PCV2 antigen by immunohistochemistry (IHC). All the pigs had serum antibodies to PCV2 and PPV at the beginning of the trial. None of them developed clinical symptoms or pathological lesions typical of post-weaning multisystemic wasting syndrome (PMWS), a disease associated to PCV2 infection. However, IHC and/or PCR analyses showed that clinically silent PCV2 infection developed in five of the eight inoculated pigs, regardless of the administration of the vaccine. In particular, PCV2 DNA and/or antigen were detected in most of the tissues examined in the two pigs with the lowest titer of maternal PCV2 antibodies at the beginning of the trial. PPV DNA was not detected in any of the samples examined. The five pigs with PCR and/or IHC evidence of PCV2 infection had a mean weight gain during the experiment lower than that of the inoculated PCR-negative pigs considered together and that of the control pigs. In conclusion, it would appear that passive immunity against PCV2 can play a role in preventing the development of PMWS, but is not able to prevent the establishing of clinically silent PCV2 infections. The dissemination and persistence of the virus in the tissues may depend on the level of PCV2 antibodies at the time of inoculation.

Experimental reproduction of postweaning multisystemic wasting syndrome (PMWS) in pigs in Sweden and Denmark with a Swedish isolate of porcine circovirus type 2.

An experimental model using 3-day-old snatch-farrowed colostrum-deprived piglets co-infected with porcine circovirus type 2 (PCV2) and porcine parvovirus (PPV) is at present one of the best methods to study factors affecting development of postweaning multisystemic wasting syndrome (PMWS). A Swedish isolate of PCV2 (S-PCV2) retrieved in 1993 from a healthy pig has been used in this model to reproduce PMWS in pigs from Northern Ireland. This virus has been present in the Swedish pig population for at least a decade without causing any known PMWS disease problems, despite its potential pathogenicity. The reasons for this are unknown, but could be related to genetics, absence of triggers for PCV2 upregulation (infectious agent and/or management forms) within Swedish pig husbandry. In order to confirm the pathogenicity of S-PCV2, Swedish and Danish pigs were experimentally infected with this isolate according to the established model. Swedish pigs were also infected with a reference isolate of PCV2 (PCV2-1010) to compare the severity of disease caused by the two isolates in Swedish pigs. Both Danish and Swedish pigs developed PMWS after the experimental infection with S-PCV2. Antibodies to PCV2 developed later and reached lower levels in serum from pigs infected with S-PCV2 than in pigs inoculated with PCV2-1010. In general, pigs infected with S-PCV2 showed more severe clinical signs of disease than pigs infected with PCV2-1010, but pigs from all PCV2-inoculated groups displayed gross and histological lesions consistent with PMWS. All pigs inoculated with PPV, alone or in combination with PCV2, displayed interleukin-10 responses in serum while only pigs infected with PPV in combination with PCV2 showed interferon-alpha in serum on repeated occasions. Thus, the pathogenicity of S-PCV2 was confirmed and a role for cytokines in the etiology of PMWS was indicated.

Porcine circovirus-2 and concurrent infections in the field.

Porcine circovirus-2 (PCV-2) is the necessary cause of post-weaning multisystemic wasting syndrome (PMWS) in swine; however, a variety of co-factors, including other infectious agents, are thought to be necessary in the full expression of disease. Porcine parvovirus (PPV) was found in the inoculum used in the first experiments to reproduce PMWS in gnotobiotic swine. Retrospective and prospective studies in the field and laboratory have demonstrated PCV-2 can act synergistically with PPV to enhance the severity of PMWS. PCV-2 has been shown to play a role in the porcine infectious disease complex (PRDC). Other co-infecting agents with PCV-2 in the lung include, porcine reproductive and respiratory syndrome virus (PRRSV), swine influenza virus (SIV) and Mycoplasma hyopneumoniae. Exposure of pregnant sows to PPV, PRRSV, or encephalomyocarditis virus may interact with PCV-2 infected foetuses. The severity of hepatic lesions in PCV-2 infected pigs may be enhanced by co-infection with agents such as swine hepatitis E virus and Aujezsky's disease virus. Additional studies are required to determine the mechanistic basis for the interaction of PCV-2 with other agents in the pathogenesis of the various clinical syndromes that have been associated with PCV-2 infection.

PMWS: experimental model and co-infections.

Porcine circovirus type 2 (PCV2) is now recognised as the causal agent of porcine multisystemic wasting syndrome (PMWS), an economically important wasting disease of young pigs [J. Vet. Diagn. Invest. 12 (2000) 3]. Gross lesions of PMWS include generalised lymphadenopathy, hepatitis, nephritis and pneumonia and typical histological lesions include lymphocytic depletion and multinucleated giant cell formation in lymph nodes, degeneration and necrosis of hepatocytes, and multifocal lymphohistocytic interstitial pneumonia. This communication will review the results of experimental infections of gnotobiotic (GN), colostrum-deprived (CD) and colostrum-fed (CF) pigs within our group, and elsewhere, with PCV2 and the conclusions that can be drawn from this work.

Postweaning multisystemic wasting syndrome--PMWS. the first year with the disease in Sweden.

Postweaning multisystemic wasting syndrome (PMWS) is causally associated with porcine circovirus type 2 (PCV2) infection of pigs. PCV2 has been present in the Swedish pig population for at least ten years. Despite this, no signs of PMWS were observed in pigs of Sweden until 2003. Since then the disease has spread slowly in Sweden. The pig population of Sweden is geographically isolated, the density of pigs and the pathogen load in the country is low and the trade of animals is organised in a restricted way. To date, there has been little information placed in the peer-reviewed literature on the study of the spread of PMWS in a country during the early phase of the disease outbreak, mainly because diagnosis of PMWS has only occurred after the disease has been recognised on a number of farms. This manuscript documents the studies to date on the PMWS outbreak in Sweden which has been closely monitored since the initial outbreak of diseases on a single farm in 2003 was diagnosed. In Sweden PMWS was officially reported for the first time at a progeny test station during December 2003. One year later sixteen herds have been diagnosed and reported to the authorities: one progeny test station, one nucleus herd, thirteen piglet producers or integrated herds and one specialised fattening herd. Fifteen suspected cases have been deemed negative for PMWS with disease scenarios associated with Lawsonia intracellularis, Brachyspira pilosicoli, Mycoplasma hyopneumoniae, shortcomings in feed composition etc. To date, one year after the index case was reported ten other herds suspected for PMWS are under observation. The PMWS cases are concentrated into the southern part of Sweden. However, the disease is spreading slowly, and in general there are no obvious physical links between affected herds. The data generated to date in Sweden suggest that introduction of a new contagious microbe into the country that is responsible for the PMWS outbreaks appears unlikely, as does the spread of PMWS via semen.

Comparison of the structures of three circoviruses: chicken anemia virus, porcine circovirus type 2, and beak and feather disease virus.

Circoviruses are small, nonenveloped icosahedral animal viruses characterized by circular single-stranded DNA genomes. Their genomes are the smallest possessed by animal viruses. Infections with circoviruses, which can lead to economically important diseases, frequently result in virus-induced damage to lymphoid tissue and immunosuppression. Within the family Circoviridae, different genera are distinguished by differences in genomic organization. Thus, Chicken anemia virus is in the genus Gyrovirus, while porcine circoviruses and Beak and feather disease virus belong to the genus CIRCOVIRUS: Little is known about the structures of circoviruses. Accordingly, we investigated the structures of these three viruses with a view to determining whether they are related. Three-dimensional maps computed from electron micrographs showed that all three viruses have a T=1 organization with capsids formed from 60 subunits. Porcine circovirus type 2 and beak and feather disease virus show similar capsid structures with flat pentameric morphological units, whereas chicken anemia virus has stikingly different protruding pentagonal trumpet-shaped units. It thus appears that the structures of viruses in the same genus are related but that those of viruses in different genera are unrelated.

Dendritic cells harbor infectious porcine circovirus type 2 in the absence of apparent cell modulation or replication of the virus.

Dendritic cells (DCs) play crucial roles in innate and adaptive immune responses, rendering them critical targets for virus infections. Porcine circovirus type 2 (PCV2) is associated with the development of postweaning multisystemic wasting syndrome (PMWS) in piglets. We demonstrate here that 80 to 90% of monocyte-derived and bone marrow-derived DCs interact with PCV2 similar to the early stages of an infection. There was no evidence for virus replication, but the virus did persist in DCs without loss of infectivity nor the induction of cell death. This could reflect an abortive infection, but there was no evidence of virus uncoating-the infectivity remained intact for at least 5 days. Alternatively, the results may reflect DC endocytosis of antigenic material. However, there was no modulation of DC surface major histocompatibility complex class I and class II, CD80/86, CD25, CD16, or CD14. Furthermore, infected DC did not transmit virus to syngeneic T lymphocytes, even when the latter were activated. Such coculture did not induce PCV2 replication or death of the lymphocytes or DCs. These results demonstrate that PCV2 can persist in DCs in the absence of virus replication or degradation. Such a silent virus infection presents a novel mechanism of not only immune evasion but also escaping the DC degradation pathway. Because of their migratory capacity, infection of DCs thus provides a potent vehicle for transport of the virus throughout the host without the need for replication. In addition, the lymphopenia seen in PMWS is not a direct effect of the virus on lymphocytes but would require additional events, as proposed by others.

In vitro studies on the infection and replication of porcine circovirus type 2 in cells of the porcine immune system.

Porcine circovirus type 2 (PCV2) nucleic acid and/or antigens are consistently observed in cells of monocytic morphology in lesions of pigs affected by post-weaning multisystemic wasting syndrome (PMWS). In this study, PCV2 antigen was detected in the cytoplasm of monocytes, pulmonary macrophages (PMs) and monocyte-derived macrophages exposed to the virus in vitro, by immunofluorescence analysis (IFA) and the phenotype of these cells confirmed by detection of monocytic cell surface markers using flow cytometry. Viral antigen was not observed in lymphocytic cells. Replication of the virus in PMs was investigated further by comparison to that observed in the continuous pig kidney cell line (PK15A) using quantitative virus titration, quantitative PCR and by the detection of double stranded DNA intermediates of viral replication by Southern blotting analyses. Although increases in viral DNA and levels of infectious virus progeny and the presence of replicative intermediates, indicative of viral replication, were observed in PK15A cells, no such changes were observed in PMs in spite of the fact that infectious virus, viral antigen and viral DNA persisted in the cells for at least the duration of the experiment. These results suggest that in vivo, monocytic cells may not represent the primary target for PCV2 replication.

Evaluation of a porcine circovirus type 2-specific antigen-capture enzyme-linked immunosorbent assay for the diagnosis of postweaning multisystemic wasting syndrome in pigs: comparison with virus isolation, immunohistochemistry, and the polymerase chain reaction.

Quantitative virus isolation, immunohistochemistry, polymerase chain reaction (PCR) assay, and a porcine circovirus 2 (PCV2)-specific antigen-capture enzyme-linked immunosorbent assay (ELISA) were used for differentiation between clinical and subclinical PCV2 infections of swine. Tissue samples from pigs experimentally infected with PCV2 and field cases of postweaning multisystemic wasting syndrome and PCV2-associated reproductive disorders were used in this evaluation. In initial studies on 6 PCV2 pools using 3 previously published PCR protocols for PCV2 detection, quantitative virus isolation, and antigen-capture ELISA, substantial differences in sensitivity were identified among these procedures. Examination of tissue samples from diseased and clinically normal pigs indicated that immunohistochemistry, quantitative virus isolation, and antigen-capture ELISA could be used to differentiate between clinical and subclinical PCV2 infections, but the PCR assay could not. Because subclinical infections of pigs with PCV2 are common, the use of nonquantitative PCR as a diagnostic tool for PCV2-related diseases should be discouraged and the PCV2-specific antigen-capture ELISA evaluated further.

Porcine circovirus 2 infection in swine foetuses inoculated at different stages of gestation.

The ability of porcine circovirus 2 (PCV2) to replicate and cause pathologic abnormalities in foetuses at selected time points of gestation was examined in this study. Two foetuses were inoculated in utero in each of two sows at 57, 75 and 92 days of gestation, respectively, with PCV2 (1121). The remaining foetuses were left uninoculated to assess whether intra-uterine spread occurred. Twenty-one days after inoculation, the foetuses were collected and examined for gross lesions and for virus and infected cells in different organs. Serum samples from all foetuses were tested for PCV2 antibodies. Virus replication was detected in all inoculated foetuses. Spread to non-inoculated foetuses did not occur. Virus replication was significantly higher in foetuses inoculated at 57 days compared to that inoculated at 75 and 92 days. The heart contained the highest virus titre and highest number of viral antigen positive cells. Gross lesions were observed only in foetuses inoculated at 57 days of age. PCV2 antibodies were detected only in foetuses inoculated at 75 and 92 days. This study shows the ability of PCV2 to replicate in foetuses at different stages of gestation and to cause pathologic abnormalities in foetuses inoculated at 57 gestational days.

Production, characterisation and applications of monoclonal antibodies to porcine circovirus 2.

The production, preliminary characterisation and applications of monoclonal antibodies (mabs) against six porcine circovirus 2 isolates are described. A total of 14 stable hybridomas were produced, of which 7 were characterised. All of the mabs characterised were of IgG isotype. All the mabs tested reacted by IIF with acetone-fixed cell cultures infected with PCV2 isolates from Canada, France, Spain, Denmark, USA and UK. No cross-reactivity with a porcine circovirus 1 field isolate was demonstrated using the panel of mabs tested. In addition, one of the seven mabs tested demonstrated neutralising activity against PCV2 isolates from Canada and France. The use of selected PCV2-specific mabs for the development of virus detection methodologies is described.

Lack of antibodies to porcine circovirus type 2 virus in beef and dairy cattle and horses in western Canada.

Porcine circovirus type 2 (PCV2) is a recently recognized agent that is consistently associated with postweaning multisystemic wasting disease in swine. There are conflicting data concerning the ability of this virus to infect and cause disease in other species. To determine if normal cattle, cattle affected with various illnesses, and normal horses in endemic areas of PCV2 infection in swine have had PCV2 infections, 100 randomly selected bovine sera, 100 equine sera, and 100 colostrum samples from clinically normal dairy cattle were examined for the presence of antibodies to porcine circoviruses by using ELISAs. All samples tested were negative for antibodies to porcine circoviruses. As well, a seronegative neonatal Holstein calf and 6 seronegative, 6-month-old beef calves that were experimentally infected with PCV2 failed to develop antibodies to the virus. These results suggest that natural infection of cattle and horses with PCV2 does not occur, or is a rare event, in western Canada.