Porcine Circovirus 2 Infection Research Articles

TLR2/MyD88/NF-κB signalling pathway regulates IL-8 production in porcine alveolar macrophages infected with porcine circovirus 2.

Porcine circovirus 2 (PCV2) is the primary cause of post-weaning multisystemic wasting syndrome, in which it stimulates a strong IL-8 response that is associated with chronic inflammation as well as lesions in the lymphoid organs. However, the mechanism underlying PCV2-induced IL-8 production is still unclear. In the present study, we demonstrated that increased IL-8 expression during PCV2 infection depends on Toll-like receptor (TLR2), but not TLR4 or TLR9 signalling pathways in porcine alveolar macrophages. Moreover, we found that impairment of the MyD88/NF-κB signalling pathway by MyD88 knockdown or NF-κB inhibitors markedly decreased PCV2-induced IL-8 secretion. These results suggest that PCV2 induces IL-8 secretion via the TLR2/MyD88/NF-κB signalling pathway. Therefore, it is important to elucidate the molecular mechanisms of the PCV2-induced inflammatory response.

Analysis of TTSuV1b antibody in porcine serum and its correlation with four antibodies against common viral infectious diseases.

BackgroundThe purpose of the present study was to evaluate the correlation between Torque teno sus virus 1b (TTSuV1b) infection and other viral infections or vaccine immunization in conventional pigs.MethodsWith overexpressed and purified viral protein TTSuV1b as antigen, an indirect enzyme-linked immunosorbent assay (ELISA) method for detecting TTSuV1b antibody was established, which demonstrated great specificity and reproducibility. Porcine serum samples (n = 212) were tested using ELISA. Meanwhile, the antibodies against Classical Swine Fever Virus (CSFV), Pseudorabies Virus (PRV), Porcine Reproductive and Respiratory Syndrome Virus (PRRSV), and Porcine Circovirus 2 (PCV2) were also examined using the commercial kits.ResultsStatistical analysis indicated that the level of anti-TTSuV1b antibody was positively correlated with the level of anti-PCV2 antibody in a lesser extent; the level of antibodies against TTSuV1b or PCV2 were significantly lower in porcine serum with low level of TTSuV1b virus, implicating the potential consistency and synchronization in the mechanism of TTSuV1b and PCV2 infection. Whereas, antibodies against PRRSV or CSFV showed no statistical significance on comparison with anti-TTSuV1b antibody, implicating that in conventional pigs, the antibody level for PRRSV and CSFV were not significantly influenced by TTSuV1b infection.ConclusionIn conclusion, examination of anti-TTSuV1b antibody in porcine serum with the presently established ELISA method would serve as a supplementary approach for etiological investigation, and the combined statistical analysis of the antibodies against four other viruses might help to further understand the TTSuV1b infection as well as its pathogenicity.

Effect of deoxynivalenol (DON) mycotoxin on in vivo and in vitro porcine circovirus type 2 infections

Deoxynivalenol (DON) is a mycotoxin produced by Fusarium spp and is a common contaminant of grains in North America. Among farm animals, swine are the most susceptible to DON because it markedly reduces feed intake and decreases weight gain. Porcine circovirus type 2 (PCV2) is the main causative agent of several syndromes in weaning piglets collectively known as porcine circovirus-associated disease (PCVAD). The objectives of this study were to investigate the impact of DON on PCV2 replication in NPTr permissive cell line, and to determine eventual potentiating effects of DON on PCV2 infection in pigs. Noninfected and infected cells with PCV2 were treated with increasing concentrations of DON (0, 70, 140, 280, 560, 1200ng/mL) and cell survival and virus titer were evaluated 72h postinfection. Thirty commercial piglets were randomly divided into 3 experimental groups of 10 animals based on DON content of served diets (0, 2.5 and 3.5mg/kg DON). All groups were further divided into subgroups of 6 pigs and were inoculated with PCV2b virus. The remaining pigs (control) were sham-inoculated with PBS. In vitro results showed that low concentrations of DON could potentially increase PCV2 replication depending on virus genotype. In vivo results showed that even though viremia and lung viral load tend to be higher in animal ingesting DON contaminated diet at 2.5mg/kg, DON had no significant effect on clinical manifestation of PCVAD in PCV2b infected animals. DON has neither in vitro nor in vivo clear potentiating effects in the development of porcine circovirus infection despite slight increases in viral replication.

Infection of porcine circovirus 2 (PCV2) in intestinal porcine epithelial cell line (IPEC-J2) and interaction between PCV2 and IPEC-J2 microfilaments.

BackgroundPorcine circovirus-associated disease (PCVAD) is caused by a small pathogenic DNA virus, Porcine circovirus type 2 (PCV2), and is responsible for severe economic losses. PCV2-associated enteritis appears to be a distinct clinical manifestation of PCV2. Most studies of swine enteritis have been performed in animal infection models, but none have been conducted in vitro using cell lines of porcine intestinal origin. An in vitro system would be particularly useful for investigating microfilaments, which are likely to be involved in every stage of the viral lifecycle.MethodsWe confirmed that PCV2 infects the intestinal porcine epithelial cell line IPEC-J2 by means of indirect immunofluorescence, transmission electron microscopy, flow cytometry and qRT-PCR. PCV2 influence on microfilaments in IPEC-J2 cells was detected by fluorescence microscopy and flow cytometry. We used Cytochalasin D or Cucurbitacin E to reorganize microfilaments, and observed changes in PCV2 invasion, replication and release in IPEC-J2 cells by qRT-PCR.ResultsPCV2 infection changes the ultrastructure of IPEC-J2 cells. PCV2 copy number in IPEC-J2 cells shows a rising trend as infection proceeds. Microfilaments are polymerized at 1 h p.i., but densely packed actin stress fibres are disrupted and total F-actin increases at 24, 48 and 72 h p.i. After Cytochalasin D treatment, invasion of PCV2 is suppressed, while invasion is facilitated by Cucurbitacin E. The microfilament drugs have opposite effects on viral release.ConclusionPCV2 infects and proliferates in IPEC-J2 cells, demonstrating that IPEC-J2 cells can serve as a cell intestinal infection model for PCV2 pathogenesis. Furthermore, PCV2 rearranges IPEC-J2 microfilaments and increases the quantity of F-actin. Actin polymerization may facilitate the invasion of PCV2 in IPEC-J2 cells and the dissolution of cortical actin may promote PCV2 egress.

Emergence of Porcine Circovirus 2 Associated Reproductive Failure in Southern India.

Incidence of unusually high numbers of stillbirths was observed at a piggery unit at the Veterinary University research farm in Tamil Nadu State of India. Systematic examination of the tissue from stillborn piglets led to the identification of presence of Porcine circovirus 2 (PCV2). Detailed analysis utilizing electron microscopy, polymerase chain reaction and sequencing confirmed the presence of PCV2 in the tissue of affected piglets. Histopathology analysis of the affected piglet tissue showed lymphoid cell depletion of lymphnodes, spleen and infiltration of liver, kidney, myocardium, etc. Retrospective examination of the morbidity and mortality history in the farm revealed high mortality in young and weanling piglets suggestive of PCV2 infection-induced diseases. This is the first report of emergence of major disease incidence in farmed swine due to PCV2 infection in India.

HMG-CoA reductase is negatively associated with PCV2 infection and PCV2-induced apoptotic cell death

We examined the role of HMG-CoA reductase (HMGCR) during porcine circovirus 2 (PCV2) infection. The results demonstrated that levels of endogenous HMGCR were not significantly different in PCV2-infected cells and mock-infected cells. However, the level of phosphorylated HMGCR, an inactivated form of HMGCR, was increased in PCV2-infected cells. Furthermore, HMGCR was upregulated by overexpression, silenced by siRNA or inactivated using its dominant-negative form in PK-15 cells. The results showed that PCV2 infection was inhibited by HMGCR overexpression, whereas it was significantly increased in HMGCR-silenced cells and HMGCR inhibitor-treated cells. Moreover, there was a robust apoptotic response at 48 h post-infection (p.i.) in HMGCR-inactivated cells, and this response was significantly greater than that observed in PK-15 cells. A modest apoptotic response was also observed in HMGCR-silenced cells. Caspase-3 activity was also analysed in PCV2-infected cells at 48 h p.i. As expected, caspase-3 activity was significantly increased in HMGCR-inactivated and -silenced cells compared with PK-15 cells. PCV2 replication was dose-dependently increased in HMGCR-inactivated cells when treated with increasing amounts of caspase-3 inhibitor. Altogether, HMGCR was negatively associated with PCV2 infection and PCV2-induced apoptotic cell death. These data demonstrated that HMGCR can be used as a candidate target for PCV2 disease control and antivirus research. Furthermore, the cells generated in this study can be used to evaluate the potential effects of HMGCR on PCV2 replication.

Primer informe de circulación de Circovirus porcino tipo 2 (PCV2) en Ecuador

Este reporte es la primera identificación de Circovirus porcino tipo 2 (PCV2) en cerdos del Ecuador. Desde noviembre 2010 a Marzo 2011 se colectaron muestras de 162 animales: 25 animales enfermos provenientes de granjas comerciales y 137 a partir de camales. Para detectar ADN de PCV2 utilizamos la reacción en cadena de la polimeraza (PCR) con dos pares de cebadores. Se detectó positividad en 24 de los 25 cerdos analizados a partir de granjas comerciales y en 77 de los 137 animales provenientes de camales. El análisis de secuencias de ADN obtenidas a partir de nueve amplicones confirmó la detección de PCV2 con al menos el 95% de homología en relación a otras secuencias de PCV2 encontradas en la base de datos del GenBank. Adcionalmente las lesiones microscópicas en los tejidos de cerdos clínicamente afectados, fueron compatibles con infección por circovirus porcino.

Induction of mucosal immunity by intranasal immunization with recombinant adenovirus expressing major epitopes of Porcine circovirus-2 capsid protein

Porcine circovirus-2 (PCV-2) is primarily transmitted through mucosa, thus the mucosal immunity may constitute an essential feature of vaccination strategies against PCV-2 infection. Mucosal immunity elicited by recombinant replication-deficient adenovirus expressing the major epitopes of PCV-2 capsid protein (rAd/Cap/518) via intranasal (i.n.), intramuscular (i.m.) or oral routes in mice were evaluated. Immunization with rAd/Cap/518 via i.n. route induced higher titers of IgA in saliva, bronchoalveolar and intestinal lavage fluid compared with those immunized via i.m. route. The proportions of CD3+, CD3+CD4+ and CD3+CD8+ T cells were significantly increased in mice immunized with rAd/Cap/518 via i.n. route compared with the control group. Higher levels of IFN-γ were detected in the spleen and mesenteric lymph nodes of mice immunized with rAd/Cap/518 via i.n. route compared with other groups, yet IL-4 was not detected in any group. Real-time PCR analysis confirmed viral DNA loads in the i.m. or i.n. immunization group was lower than that seen in the rAd immunization. These results indicate that i.n. administration of rAd/Cap/518 can elicit humoral and Th1-type cellular protective immunity in both systemic and mucosal immune compartments in mice, representing a promising mucosal vaccine candidate against PCV-2.

Dynamics of antibody response and viraemia following natural infection of porcine circovirus 2 (PCV‐2) in a conventional pig herd

Porcine circovirus 2 (PCV-2) is a primary agent of post-weaning multi-systemic wasting syndrome (PMWS), ubiquitous in pig herds. The course of viraemia and seroconversion in naturally infected pigs were investigated in piglets from the 2nd week of their life. Piglets were divided into seropositive (Ab(+)) and seronegative (Ab(-)) groups. Subsequently, after vaccination against PCV-2 (Ingelvac(®) CIRCOFLEX™, Böehringer Ingelheim), they were further divided into non-vaccinated seronegative (NVAC/Ab(-)) and seropositive (NVAC/Ab(+)), and vaccinated seronegative (VAC/Ab(-)) and seropositive (VAC/Ab(+)). PCV-2 colostral antibodies failed to prevent development of natural PCV-2 infection in conventional piglets; however, this occurred at a higher age in comparison with seronegative pigs. Neither colostral nor post-infection antibodies prevented development of viraemia, which persisted up to the end of the study (the 19th week), but without clinical signs of PMWS. Vaccination failed to prevent development of natural PCV-2 infection, but viraemia was limited to between the 8th and 10th week. The presence of colostral anti-PCV-2 antibodies did not show any untoward effect to vaccination; on the contrary, VAC/Ab(+) animals showed the lowest titre of viraemia.

Comparative analysis of different methods to enhance porcine circovirus 2 replication

Porcine circovirus 2 (PCV2) is an extremely slow-growing virus, and PCV2 infection and replication in cell culture yield very low viral titers. The effects of different methods of PCV2 cultivation in vitro were compared with the purpose of increasing viral yield. The results showed that treatment with IL-2, ConA, and D-glucosamine increased PCV2 yield more effectively than other treatments. Additionally, treatment with IL-2, ConA, D-glucosamine and MβCD consistently increased PCV2 infection in PK-15 cells during consecutive viral passages. A combinatorial treatment with ConA, MβCD and D-glucosamine increased PCV2 yield significantly in PK-15 cells, to 1.81×10(10) genome copy numbers per mL of cell lysate at 72 hpi, and the viral titer (-lgTCID50/100 μL) was 8.6. The results of this study may be helpful for the investigation of PCV2 replication and the production of a PCV2 vaccine.

Verification of natural infection of peridomestic rodents by PCV2 on commercial swine farms

The porcine circovirus-2 (PCV2) is the main agent responsible for porcine circovirus associated diseases (PCVAD). Few studies have been done regarding PCV2 infection in other species. The purpose of this study was to investigate the occurrence of PCV2 infection in the peridomestic rodent species Mus musculus and Rattus rattus on commercial pig farms in Brazil. Immunohistochemistry assay demonstrated PCV2 in the spleen, lung and kidney. Viral DNA was detected in tissues by nested PCR assay. Partial sequences of PCV2 genomes detected in the rodents had strong identity with gene sequences of PCV2 isolates from pigs. These results show that the studied peridomestic rodent species can be naturally infected by PCV2. However, further studies are needed to confirm PCV2 transmission from rodents to pigs.

Lack of antibody protection against Porcine circovirus 2 and Porcine parvovirus in naturally infected dams and their offspring

The aim of this study was to evaluate Porcine parvovirus (PPV) and Porcine circovirus 2 (PCV2) infection in dams and their offspring and the role of antibody protection on these infections. Sera were collected from gilts and sows by venipuncture and from umbilical cord of newborn pre-suckle piglets for the detection of PCV2 and PPV antibodies by immunoperoxidase monolayer and haemmaglutination inhibition assays, respectively. Gilts and sows sera were submitted to viral detection by PCR, as well as heart, lung, tonsil and lymph nodes samples from stillborn and mummified fetuses. High antibody titers before artificial insemination (AI) (>5.120 and >2.560 UHA for PCV2 and PPV, respectively), were found associated with viremia and fetal exposure for both PCV2 and PPV, respectively, in gilts and sows, regardless of pregnancy stage. These infections resulted in litters with mummified, stillborn, as well as seropositive and viable newborns. These findings bring new evidence about the lack of antibody protection against PCV2 and PPV infections in dams, indicating that more studies are necessary about the role of humoral response against both pathogens.

Subclinical porcine circovirus infection: What lies beneath

Subclinical porcine circovirus infection: What lies beneath

Porcine Circovirus 2: Immunopathogenesis and Recent Developments in Vaccines

Porcine circovirus 2 (PCV2) is currently considered an important etiologic agent of swine and its infection has potentially serious economic impact on the swine industry worldwide. This virus is frequently associated with postweaning multisystemic wasting syndrome (PMWS), and also with other clinical conditions such as porcine dermatitis and nephropathy syndrome (PDNS), late-term abortions, reproductive failure in sows, proliferative and necrotizing pneumonia and congenital tremors. The term porcine circovirus-associated disease (PCVAD) is currently used to refer to any of these diseases when they are associated with PCV2 infection. The PCV2 was recognized as a pathogen in 1997, and many questions regarding its biology and pathogenesis remain unanswered. Currently, some studies have shown the production of new vaccine candidates and field efficacy testing of commercial vaccines. This review discusses some major points concerned with immunopathogenesis and vaccines for PCV2 infection.

Clinical manifestations associated with the complex circovirosis swine in the state of Goiás, Brazil

The quality attributes of the diseases present large variations, which in turn, influence the apparent signs of the animal. This report demonstrates that the clinical signs of porcine circovirus infection swine and associated diseases are present in the State of Goiás, being the respiratory signs those of higher frequency and greater responsible for the increase in production costs, since they raise the costs of treatments to control of concomitant diseases or those related to the respiratory complex The quality attributes of the diseases present large variations, which in turn, influence the apparent signs of the animal. This report demonstrates that the clinical signs of porcine circovirus infection swine and associated diseases are present in the State of Goiás, being the respiratory signs those of higher frequency and greater responsible for the increase in production costs, since they raise the costs of treatments to control of concomitant diseases or those related to the respiratory complex.

Qualitative and quantitative distribution of PCV2 in wild boars and domestic pigs in Germany

Porcine circovirus 2 (PCV2), the causative agent of postweaning multisystemic wasting syndrome (PMWS), has been detected in North American and European wild boars at prevalences arguing for high circulation rates among populations. Systematic data on the qualitative distribution of PCV2 infections and on PCVD (PCV2 diseases) in wild boars are rare, however, and quantitative data about viral loads are missing. To be able to judge the PCV2/PCVD situation in wild boars, evaluation of the nationwide qualitative and quantitative distribution of PCV2 and PCVD in Germany was the objective of the present study. Wild boar samples were compared with domestic pig samples of the same greater areas, including tonsils, lungs, spleen, Lnn. bronchiales and Lnn. mesenterici of 349 wild boars and 348 domestic pigs. All of the wild boars and 308 of the domestic pigs have been apparently free of PCVD, 40 of the domestic pigs had been rejected from slaughter due to health problems (i.e. wasting). Tissues were examined by pathohistology, immunohistology (IHC), nested PCR (nPCR and quantitative PCR (qPCR). One wild boar (0.3%) and 8.7% of the domestic pigs were classified as PCVD-affected, based on pathohistology and IHC. PCV2 DNA was detected in 63.1% and 45.4% of the wild boars by nPCR and qPCR, respectively, and in 100% and 98.8% of the domestic pigs. PCV2 loads differed significantly between wild boars (average: 10 2.8 PCV2 genomes/μg extracted sample DNA) and domestic pigs (average: 10 4.2 PCV2 genomes/μg of sample DNA). The qualitative detection of PCV2 DNA in tissues of wild boars and domestic pigs was abundant and not of any pathological relevance. The overall load of PCV2 in domestic pigs was relatively high and borderline with respect to PCVD, and there was no difference between apparently healthy pigs and pigs rejected from slaughter in this respect. Most of the wild boars were infected with PCV2 at loads less relevant for PCVD.

Colostral transmission of porcine circovirus 2 (PCV-2): reproduction of post-weaning multisystemic wasting syndrome in pigs fed milk from PCV-2-infected sows with post-natal porcine parvovirus infection or immunostimulation

Post-weaning multisystemic wasting syndrome (PMWS) was reproduced in pigs fed colostrum and milk from porcine circovirus 2 (PCV-2)-infected sows and infected post-natally with porcine parvovirus (PPV) or immunostimulated. Pregnant sows were inoculated intranasally with either PCV-2 (n=5) or PCV-2-free PK-15 cell lysates (control, n=10) 3 weeks before the expected farrowing date. Newborn piglets from five of the control sows were introduced to PCV-2-infected sows (n=6 for each sow) and allowed to feed on the colostrum for 12 h and then given 15 ml milk five times a day for 7 days. Newborn piglets from the other five control sows were fed colostrum and milk from their own sows. After 7 days, two piglets from each group were randomly selected to confirm PCV-2 infection. Twenty-one pigs fed by PCV-2-infected sows were randomly divided into three groups and subjected to post-natal PPV infection (group 1), immunostimulation (group 2) or no post-natal treatment (group 3). Twenty-one pigs fed by uninfected sows were also randomly divided and subjected to post-natal PCV-2 and PPV infection (group 4), post-natal PCV-2 infection (group 5) or no treatment (group 6, negative control). Body weight was significantly greater in group 6 than in groups 1, 2 and 4 at 49, 52, 56, 59 and 63 days of age. The typical granulomatous inflammatory reaction and lymphoid depletion of PMWS was observed in the lymph nodes of groups 1, 2 and 4 at 63 days of age. Group 3 had significantly fewer PCV-2-positive cells than groups 1, 2 and 4. In conclusion, PCV-2 shed from colostrum and milk is infectious and reproduces PMWS with post-natal PPV infection or immune stimulation.

Exploratory Epidemiological Study on Porcine Circovirus Type 2 Infection and Postweaning Multisystemic Wasting Syndrome in the Czech Republic

The objective of our study was to diagnose the postweaning multisystemic wasting syndrome (PMWS) and to determine the prevalence of the disease in 33 swine herds in the Czech Republic using the results of laboratory examinations of 100 pigs expressing the signs of wasting at the end of 2007. Microscopic lesions associated with the presence of porcine circovirus 2 (PCV2) antigen were detected in the lymph nodes from 39 of 100 diseased pigs (39%). Based on individual assessment of severity of microscopic lymphoid lesions associated with high amounts of PCV2 antigen, PMWS was confirmed in 4 out of 39 pigs originating from 3 of 33 herds (9%). The epidemiological study indicates that PCV2 infections associated with PMWS disease are only sporadically present in the Czech Republic. Subsequently used real time PCR technique confirmed the relation between PMWS status at the individual pig level and PCV2 DNA concentration. PCV2 DNA load in lymph nodes of PMWS-affected pigs were about 3 logs higher than the levels detected in the PMWS-nonaffected group (P < 0.05). Other parallel viral infections (PRRSV, PPV) were detected by real time PCR techniques in 21 out of 39 PCV2 infected pigs (54%). The results of serological examination of blood samples collected during the necropsy of 100 pigs are suggestive of great prevalence of PCV2 infections in pig herds; nevertheless serum samples collected from individual pigs at a single point in time had a low diagnostic value.

Systemic Toxoplasmosis and Concurrent Porcine Circovirus-2 Infection in a Pig

SummarySystemic toxoplasmosis and concurrent infection with porcine circovirus-2 (PCV-2) was diagnosed in a fattening pig. Clinical examination of the herd showed that up to 30% of the pigs of this weight group suffered from severe respiratory signs including sneezing and coughing, with a mortality rate of up to 5%. Gross necropsy examination revealed severe interstitial pneumonia and generalized lymphadenopathy. On microscopical examination there was necrotizing inflammation of the lung, adrenal glands and lymph nodes, associated with lymphoid depletion, cytoplasmic basophilic botryoid inclusion bodies and protozoal microorganisms. Infection with Toxoplasma gondii was confirmed by immunohistochemistry (IHC). Polymerase chain reaction analysis, in-situ hybridization and IHC confirmed systemic PCV-2 infection. These findings, associated with the respiratory signs and lesions in lymphoid tissues, are characteristic for post-weaning multisystemic wasting syndrome (PMWS). In this case, immunosuppression by PCV-2 may have triggered systemic toxoplasmosis, or immune stimulation caused by coinfection with T. gondii may have caused extensive replication of PCV-2.

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.