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Co-infection by a type 1 modified live vaccine-like strain (MLV1-like) of porcine reproductive and respiratory syndrome virus (PRRSV) and a type 2 porcine circovirus (PCV2) was identified on a French pig farm with post-weaning multisystemic wasting syndrome (PMWS). An in vivo experiment was set up to characterize the virulence level of the MLV1-like strain compared with the parental MLV1 strain, and to assess the impact of PCV2 co-infection on the pathogenicity of both PRRSV strains. Six groups of six pigs each were inoculated only with either one of the two PRRSV strains or with PCV2, or co-inoculated with PCV2 and MLV1 or PCV2 and MLV1-like strains. Six contact pigs were added to each inoculated group to assess viral transmission. The animals were monitored daily for 35 days post-inoculation for clinical symptoms. Blood and nasal swabs were sampled twice a week, and tissue samples were collected during necropsy for viral quantification. Compared to MLV1-infected pigs, animals infected with the MLV1-like strain had increased PRRSV viremia and nasal shedding, a higher viral load in the tonsils, and lymph node hypertrophy at microscopic level. PCV2 co-infection did not influence clinical, virologic or transmission parameters for MLV1, but co-infected MLV1-like/PCV2 pigs had the most severe lung lesions, the highest viremia in contact animals and the highest transmission rate. Our study demonstrated that the MLV1 strain tested was safe when co-inoculated with PCV2 in piglets. However, co-infection by the MLV1-like strain and PCV2 resulted in increased virulence compared with that due to a single infection., Competing Interests: Declaration of Competing Interest The authors declare that they have no conflict of interests., (Copyright © 2020 Elsevier B.V. All rights reserved.)

This study was designed to assess the diagnostic characteristics of two PCV2 ELISAs without a gold standard. Four hundred and sixty-five serum samples from finishing pigs (25 herds) not vaccinated against PCV2 were used. Samples were tested by two ELISAs: an in-house ELISA (I-ELISA) and the commercial SERELISA ® PCV2 Ab Mono Blocking kit (S-ELISA). A ROC curve was used to assess the S-ELISA's optimal threshold by taking the I-ELISA as a reference and using the cut-off previously determined by comparison to an cccmonolayer assay (IPMA). This led to an S-ELISA result ≥170 being considered as positive. The sensitivity (Se) and specificity (Sp) of each ELISA were then estimated without a gold standard using a Bayesian approach. The mean Se and Sp values of the I-ELISA were slightly higher than those of the S-ELISA (mean Se I-ELISA=0.90 vs. mean Se S-ELISA=0.86; mean Sp I-ELISA=0.92 vs. mean Sp S-ELISA=0.85). However, the 95% credibility intervals (CI95%) overlapped (Se I-ELISA CI95%=0.85-0.95 vs. Se S-ELISA CI95%=0.82-0.90; Sp I-ELISA CI95%=0.82-0.98 vs. Sp S-ELISA CI95%=0.75-0.94). Both ELISAs appeared to be valuable tools for detecting PCV2 antibodies., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Porcine circoviruses (PCV) are small, non-enveloped single-stranded DNA-viruses. Porcine circovirus type 2 (PCV-2) is the causal agent of post-weaning multisystemic wasting syndrome (PMWS) whereas porcine circovirus of type 1 (PCV-1) is non- pathogenic. gC1qR is a membrane-located receptor of the complement protein subunit C1q and interacts with PCV capsid proteins. The mechanisms associated with the triggering of PMWS are not well known and gC1qR may have a role in the life cycle and eventually in the pathogenicity of PCV. The objectives of this study were to determine the level of expression of gC1qR during early PCV-2 infection, to determine the region of PCV-2 capsid protein (Cap) required for the interaction with gC1qR and to evaluate the interaction of gC1qR with Cap proteins of different PCV strains. The results indicate that gC1qR transcripts are downregulated in the tonsils and the tracheo-bronchial lymph nodes of piglets infected by PCV-2 at the early time of the infection. The N-terminal amino acids (a.a. 1-59) of PCV-2b Cap, an arginine rich region, are involved in the interaction with gC1qR. Porcine gC1qR interacts with Cap proteins of two pathogenic viral strains, PCV-2a and PCV-2b, while interaction has been observed with only one Cap protein of two investigated strains of PCV-1. The amino acids 30 and 49 of PCV-1Cap, solely, were not responsible of the difference of interaction observed. We have also shown that gC1qR interacts strongly with PCV-2Caps and PCV-1 GER Cap. This result suggests that the different interaction of gC1qR with PCV Cap proteins may have an impact on the pathogenicity of the PCV., (Copyright © 2016 Elsevier B.V. All rights reserved.)

The porcine circovirus type 2 (PCV-2) is associated with several diseases including reproductive failure. This syndrome has been experimentally reproduced twice with two PCV-2 isolates representative of each major PCV-2 genogroup, i.e. PCV-2a and PCV-2b (Cariolet et al., 2002; Rose et al., 2007). In these two previous studies, the sows were infected by intra-uterine inoculation at insemination with 10(4.3) and 10(3.18) TCID(50) of PCV-2a and PCV-2b, respectively, corresponding to 1.2 × 10(11) and 3 × 10(10) genome copies, respectively. The aim of this present study was to quantify viral shedding in semen from specific-pathogen-free (SPF) boars infected with isolates from the two major PCV-2 genogroups a and b. We studied the transmission of the PCV-2 virus through contaminated semen to SPF sows and their offspring. The four inoculated boars developed sub-clinical PCV-2 infections and PCV-2 genomes were occasionally detected in semen after nasal infection of boars, with up to 1.2 × 10(6)copies/mL in the sperm-rich fraction. When PCV-2-contaminated semen was inoculated in SPF sows at artificial insemination, the sows and their offspring did not show any signs of PCV-2 infection or PCV-2 antibodies or genomes. In the present study, sows were inoculated with a maximal dose of 1.7 × 10(7) viral genome copies, which is lower than the genomic loads (i.e. 1.2 × 10(11) and 3 × 10(10) genome copies) that have been shown to induce reproductive troubles in intra-uterine inoculated sows. Our results together with the previous experiment findings suggest that PCV-2-induced reproductive disorders depend on the infectious dose inoculated to sows by the intra-uterine route., (Copyright © 2012 Elsevier B.V. All rights reserved.)

A plasmid rendered replicative in mammalian cells by inserting the Porcine circovirus 2 (PCV2) origin of replication and replicase gene (Ori-rep) has been previously constructed. The aim of the present study was to evaluate if the replication capacity of this plasmid could be advantageously used to improve the protective immunity induced by DNA vaccination. In this case we used the porcine Pseudorabies virus (PrV) DNA vaccination model. The replicative capacity of the DNA vaccine did not improve the protective immunity against PrV in pigs, but on the contrary the presence of the PCV2 Ori-rep sequence was harmful in the induction of this immunity compared to an equivalent but non-replicative DNA vaccine. In addition, the distribution and the persistence of the replicative and non-replicative plasmids inside the body were the same. This is the first study showing an in vivo deleterious effect of the replicative active PCV2 Ori-rep on the natural and specific protection against PrV infection., (Copyright © 2012 Elsevier B.V. All rights reserved.)

A study was carried out in 125 farrow-to-finish pig herds to assess the relationships between pathogens involved in respiratory disorders and to relate these findings to clinical signs of respiratory diseases and pneumonia and pleuritis at slaughter. Clinical examination and sampling were carried out on four different batches in each herd (pigs aged 4, 10, 16 and 22 weeks). Mycoplasma hyopneumoniae, Actinobacillus pleuropneumoniae, swine influenza viruses (SIV), porcine reproductive and respiratory syndrome virus (PRRSV) and porcine circovirus type 2 (PCV2) were detected by serological or PCR tests. Pneumonia-like gross lesions and pleuritis were scored at the slaughterhouse. The results indicate that the percentage of pigs PCR-positive for PCV2 at 4, 10 and 16 weeks old was associated with the percentage of pigs PCR-positive for M. hyopneumoniae at these ages. On the other hand, the percentage of pigs with antibodies against PRRSV at 10, 16 and 22 weeks was positively correlated with the percentage of pigs seropositive for M. hyopneumoniae at 22 weeks, with the percentage of pigs with antibodies against SIV H1N1 and SIV H1N2 and the percentage of pigs sero-positive for A. pleuropneumoniae serotype 2. The findings also indicate that, within the five studied pathogens, M. hyopneumoniae, PRRSV and SIV H1N1 are the major pathogens involved in pneumonia-like gross lesions even though PCV2 may play a role. A. pleuropneumoniae serotype 2, in association with PRRSV, is significantly associated with extensive pleuritis. Respiratory diseases could be significantly reduced by implementing measures including appropriate management practices to control these pathogens., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Porcine circovirus type 2 (PCV-2) is involved in several diseases named porcine circovirus-associated diseases and is transmitted by oro-faecal route. In this study we inoculated porcine-circovirus free piglets by mucosal routes (intratracheal or oro-nasal routes) with a plasmid carrying two copies of PCV-2 genomic DNA and compared the results to the intramuscular route. We observed that this PCV-2 naked DNA serves as template for viral replication and infectious PCV-2 particles are detected in the whole body after parenteral (intramuscular) or mucosal (intratracheal or oro-nasal) delivery. These results suggest that PCV-2 genome could play a role in in vivo transmission., (Copyright © 2012 Elsevier B.V. All rights reserved.)

PCV2 has been highly prevalent in the pig population for decades, prior to the emergence of associated clinical disease manifestations that severely affected the pig production worldwide in the late 90s. PCV2 can be further subdivided into several genotypes. From descriptive epidemiologic data, there is evidence of a global shift of the main PCV2 genotypes in different countries from PCV2a to PCV2b, which is generally associated with more severe disease. In addition, from analytic epidemiologic studies, the modified within-herd PCV2 dynamics of infection is strongly related to the increased incidence of clinical disorders associated with PCV2 infection. Because PCV2 is shed for a long time by an extremely large variety of routes, it easily spreads within the population both through horizontal and vertical transmission. Even if airborne transmission cannot be formally excluded, direct contact is certainly the most efficient infectious route due to the simultaneous exposure of susceptible pigs to contaminated respiratory, digestive, and urinary secretions since the probability of transmission is strongly limited by the distance between infectious and susceptible animals. Consequently, farm to farm transmission is restricted to the introduction of infected animals or infected animal products such as semen. More information would be required to assess the risk of other vehicles such as vaccines or feed ingredients since the probability of these products to be contaminated by PCV2 is unknown. However, owing to its transmission characteristics, PCV2 is able to be maintained within pig farms for years without any further need for re-introduction due to the population dynamics of modern pig operations, which continually renew the pool of the susceptible population through replacements and pig movements between compartments., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Porcine circovirus type 2 (PCV-2) is the causal agent of the post-weaning multisystemic wasting syndrome (PMWS). PCV-2 are small single-stranded circular DNA viruses clustered into two main genogroups: PCV-2a and PCV-2b. Each genogroup present a specific highly-conserved motif of six amino acids (between amino acids 86 and 91) in the PCV-2 capsid protein. The aim of this study was to verify whether the motif located in the capsid protein and specific to each PCV-2 genogroup contributes to virulence. Two parental DNA clones, PCV-2a and PCV-2b, were constructed as well as two mutants DNA clones, PCV-2a/motif 2b and PCV-2b/motif 2a by exchanging the capsid motif of each genogroup. The four DNA clones were characterized in vitro as well as in vivo. Cells transfected by the four DNA clones produced infectious viruses. In specific-pathogen-free piglets transfected by the four infectious DNA clones, PCV-2b/motif 2a virulence was not attenuated while the PCV-2a/motif 2b virulence was drastically reduced compared to their parent virulence. These results suggest that the amino acids between positions 86 and 91 of the capsid protein are determinant for the virulence of isolates. However, the environment of this motif seems also involved.

We assessed, using a modelling approach, the influence of several management practices within a farrow-to-finish farm on the age of PCV-2 infection. The impact of PCV-2 vaccination with different vaccination schemes on infection dynamics, was also tested. A stochastic individual-based model describing the population dynamics in a typical French farrow-to-finish pig farm was built and coupled with an epidemiological model of PCV-2 infection. The parameters of the infectious model were mainly obtained from previous transmission experiments. Results were subjected to a survival analysis of time-to-infection. For each comparison, the reference situation was no vaccination followed by random mixing of piglets after birth and after weaning. The risk of early infection was significantly reduced when mixing of piglets was reduced at different stages (avoiding cross-fostering and grouping piglets by litters in small pens after weaning, hazard ratio (HR)=0.52 [0.46; 0.59]). Sow-targeted vaccination delayed the infectious process until the waning of passive immunity and piglet-targeted vaccination considerably decreased the force of infection leading to a dramatic decrease of the total number of infections (HR=0.44 [0.37; 0.54]). The effect was even more pronounced when strict management measures were applied (HR=0.24 [0.19; 0.31]). Changing from a low (3%) prevalence of PCV-2-infected semen to a higher one (18%) significantly increased the risk of early infections (HR=1.36 [1.2; 1.53]), whereas reducing replacement rate or changing sow housing from individual crates to group housing had a limited impact on PCV-2 dynamics.

Porcine circoviruses are circular single-stranded DNA viruses that infect swine and wild boars. Two species of porcine circoviruses exist. Porcine circovirus type 1 is non pathogenic contrary to porcine circovirus type 2 which is associated with the disease known as Post-weaning Multisystemic Wasting Syndrome. Porcine circovirus DNA has been shown to replicate by a rolling circle mechanism. Other studies have revealed similar mechanisms of rolling-circle replication in plasmids and single-stranded viruses such as Geminivirus. Three elements are important in rolling-circle replication: i) a gene encoding initiator protein, ii) a double strand origin, and iii) a single strand origin. However, differences exist between viruses and plasmids and between viruses. Porcine circovirus replication probably involves a "melting pot" rather than "cruciform" rolling-circle mechanism.This review provides a summary of current knowledge of replication in porcine circoviruses as models of the Circovirus genus. Based on various studies, the factors affecting replication are defined and the mechanisms involved in the different phases of replication are described or proposed.

Six successive transmission trials were carried out from 4 to 39 days post inoculation (DPI) to determine the features of the infectious period for PCV2-infected pigs. The infectiousness of inoculated pigs, assessed from the frequency of occurrence of infected pigs in susceptible groups in each contact trial, increased from 4 to 18 DPI (0, 7 and 8 infected pigs at 4, 11 and 18 DPI, respectively) and then decreased slowly until 39 days post infection (4, 2 and 1 pigs infected at 25, 32 and 39 DPI, respectively). The estimated time-dependent infectiousness was fitted to three unimodal function shapes (gamma, Weibull and lognormal) for comparison. The absence of infected pigs at 4 DPI revealed a latency period between 4 and 10 DPI. A sensitivity analysis was performed to test whether the parametric shape of the transmission function influenced the estimations. The estimated time-dependent transmission rate was implemented in a deterministic SEIR model and validated by comparing the model prediction with external data. The lognormal-like function shape evidenced the best quality of fit, leading to a latency period of 8 days, an estimated basic reproduction ratio of 5.9 [1.8,10.1] and a mean disease generation time of 18.4 days [18.2, 18.5].

Post-weaning multisystemic wasting syndrome (PMWS) and other porcine circovirus type 2 (PCV2)-related diseases have been reported throughout the world for about 10 years. The present paper reviews the knowledge acquired in different fields and is largely based on the authors' experience. The horizontal transmission of PCV2 is widely documented. Contact between pigs is the main route of transmission for both PCV2 and PMWS. However, experimental inoculation of PCV2 to pigs does not give consistent results and severe clinical signs as encountered in the field are rarely obtained. It is thus acknowledged that additional conditions are required for the disease to be severe in growing pigs. These are not all known but co-infections are thought to act as triggers. The spread of such triggers/enhancers, which may or may not be infectious, could have played a role in PMWS dissemination via normal national and international trade, in some cases conferring an epidemic pattern to this spread. Most of the risk factors identified in surveys relate to poor biosecurity and inadequate hygiene/husbandry/herd management. The good correlation between viral burden in the tissues and disease severity emphasized the role of infection pressure. Genomic analysis showed great similarities between PCV2 isolates. However, although two main genotypes (genogroups) could be distinguished from the phylogenic trees, and changes with time, no clear relationship with strain virulence was apparent. Isolates detected in PMWS-positive pigs could also be detected in healthy pigs from healthy farms. A strong sow effect was observed in disease expression in the offspring. Colostrum composition and colostrum intake are supposed to be key components of disease expression. Medication is relatively inefficient as a control measure. Commercial PCV2 vaccines are now becoming available. However, losses as a result of PMWS and PCV2-related diseases are greatly reduced by applying appropriate hygiene and husbandry practices.

PCV-2 within- and between-pen transmission was quantified by estimating the daily transmission rate beta and the basic reproduction ratio (R(0)) using a stochastic SEIR (Susceptible, Exposed, Infectious, Removed) model fitted on experimental data. Within-pen transmission was quantified by using four groups of eight SPF (specific pathogen-free) pigs (four infected and four susceptible pigs having direct contact). Between-pen transmission was studied in two groups of 16 SPF pigs (eight infected and eight susceptible pigs having indirect contact (10 cm distance)). Pigs were monitored twice a week (blood samples) and were tested for PCV-2 antibodies (ELISA test) and viral genome load in sera (real-time PCR). Transmission parameters beta(within) and beta(between) were estimated using a maximum likelihood method and the duration of infectiousness, to compute R(0), was estimated with a parametric survival model. Different assumptions were made to determine the end of infectiousness (seroconversion, seroconversion and decline in viral genome load, permanent infectiousness). R(0[within]) (8.9 (5.1-15.4)) was greater when the end of infectiousness was assumed to be related to both seroconversion and a decline of PCV-2 genome load in sera (average duration of infectiousness = 32 days) compared with only seroconversion as the indicator of recovery (R(0[within]) = 5.5 (3.3-9.0)). Whatever the assumption, between-pen R(0) (0.58 (0.23-1.47)) was always significantly lower than within-pen R(0). Only beta(within) was sensitive to the assumption on end of infectiousness and decreased with increasing duration of infectiousness. These results showed that PCV-2 transmission is influenced by contact structure that appears worth being taken into account in an epidemic model.

The effect of different Parvovirus+Erysipelas vaccination schemes in PCV2-infected sows on PMWS outcome in the offspring was investigated under experimental conditions. Six PCV2-free sows were first infected oro-nasally with PCV2 two months before insemination (D0; "Day 0") and then by the intra-uterine route at insemination (D62). On D21 and D42, vaccinated sows received either the two commercial monovalent vaccines, A1(PPV) and A2(Erysipelas), or the bivalent vaccine B (PPV+Erysipelas). In addition, three SPF sows (foster-sows) were synchronized for farrowing dates to enable them to foster piglets born to infected sows and removed at birth before colostrum intake. A significantly higher proportion of mummified fetuses was obtained from PCV2-infected non-vaccinated sows than from vaccinated sows. Acute myocarditis lesions were found in their piglets, together with a high PCV2 genome load. The latter was significantly higher than in those born to PCV2-infected vaccinated sows. Sentinel PCV2-negative piglets, born to SPF foster-sows, seroconverted at almost the same time as piglets without PCV2 passive immunity and born to infected sows. Sixteen of the 84 liveborn piglets born to infected sows and foster-sows were affected by a syndrome possibly related to PMWS, as judged by clinical signs and histological lesions. Most were born to PCV2-infected non-vaccinated sows and 12/16 did not receive PCV2 passive immunity. The probability of PCV2 infection and the number of PCV2 genome copies per gram of tissue were significantly increased in piglets that did not receive PCV2 passive immunity.

Postweaning multisystemic wasting syndrome (PMWS) is a recently emerged disease affecting pigs. Type 2 porcine circovirus (PCV2) has been associated with this syndrome although other factors are required in association with this virus for PMWS expression. The aim of this study was to investigate whether general immunostimulation (injections of keyhole limpet hemocyanin emulsified in incomplete Freund adjuvant and of thioglycollate medium) could strengthen the severity of PMWS in six-week-old specific-pathogen-free (SPF) piglets transfected with pure tandem-cloned PCV2 DNA by the intramuscular route. Non-immunostimulated piglets transfected with the viral clone did not present clinical signs but only mild pathological microlesions characteristic of PMWS. These piglets seroconverted and high viral genome loads and infectious titers were detected in the lymphoid organs at the end of the trial. Mild-to-moderate forms of PMWS were generally observed in the immunostimulated transfected piglets, as well as one severe form for a piglet (8003) which died. These piglets with mild-to-moderate forms had higher DNA loads than the transfected-only animals. Thus, viral replication was enhanced by immunostimulation. This is the first time that clinical PMWS has been reported in an SPF immunostimulated piglet infected with a pure inoculum consisting of tandem-cloned PCV2 DNA. This result confirms that PCV2 is the agent of PMWS and that immunostimulation could enhance PMWS in SPF piglets transfected with a PCV2 DNA clone.

Post-weaning multisystemic wasting syndrome (PMWS) is a complex disease syndrome in swine, affecting nursery and fattening pigs. Although ongoing evidence suggests that porcine circovirus type-2 (PCV2) is the causal agent of PMWS, the host immune system appears to have a crucial role in the PMWS pathogenesis of PCV2-affected pigs. Owing to difficulties in producing a biologically pure form of PCV2 devoid of the other viral agents commonly present in swine tissues, we decided to use a tandem-cloned PCV2 DNA providing highly pure grade reagent in order to monitor the virulence of PCV2 alone or with an immunostimulating co-factor, granulocyte-macrophage colony-stimulating factor (GM-CSF). A single intramuscular injection of tandem-cloned PCV2 DNA into 5-week-old piglets produced plasmid to viral genome progeny and infectious particles as early as 8 days post-injection in all the organs tested (the lung, the tonsil and the inguinal, mesenteric, bronchial and upper-right axial lymph nodes). The initial plasmid load was not detected with the help of primers designed to specifically detect the acceptor plasmid, thus confirming the replication of the viral genome. Despite the presence of a high level of PCV2 genome copies in the lymphoid organs--the tonsil and the lung--and the presence of infectious particles, no detectable clinical manifestations or pathological lesions were observed in the transfected pigs over the period of observation, regardless of whether they had been co-injected with plasmid containing GM-CSF DNA or had received plasmid containing PCV2 DNA alone. GM-CSF encoding DNA injection had no significant effect on viral replication or on the production of viral particles and appearance of the disease.