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Abstract Background The immunocrit is a cost-effective and straightforward technique traditionally used to assess passive immunity transfer to newborn piglets. However, it has not been previously used for monitoring the effect of vaccination and/or infections. Therefore, this study aimed to evaluate the usefulness of the immunocrit technique as an immunological monitoring tool in a vaccination and challenge scenario, using porcine circovirus 2 (PCV-2) as pathogen model. The immunocrit ratio was monitored in PCV-2 vaccinated (V) and non-vaccinated (NV) 3-week-old piglets (study day 0, SD0) that were subsequently challenged with this virus at SD21 and followed up to SD42. Additional techniques (PCV-2 IgG ELISA, optical refractometry, and proteinogram) were performed to further characterize the results of the immunocrit analysis. Results Immunocrit, γ-globulin concentration and PCV-2 S/P values followed similar dynamics: descending after PCV-2 vaccination but ascending after an experimental PCV-2 inoculation. However, statistically significant differences between V and NV animals were only found with the PCV-2 ELISA. In this case, V animals had significantly higher (p

Porcine circovirus type 2 (PCV2) is the main and primary causative agent of Postweaning Multisystemic Wasting Syndrome (PMWS). To date, immunoperoxidase monolayer assay (IPMA), indirect immunofluorescent assay (IFA), and enzyme linked immunosorbent assay (ELISA) are the most commonly diagnostic methods for detecting PCV2 antigens. However, these methods require specialized equipment and technical expertise and are suitable for laboratory use only. This study aims to develop an immunochromatographic strip and a magnetic chemiluminescence immunoassay for the detection of PCV2 antigens. The recombinant protein was constructed using a prokaryotic expression system, and the polyclonal antibody was obtained by animal experiments. Polystyrene microspheres are used as solid phase carriers to covalently bind to the amino groups of proteins to form immunoprobes. Monodisperse beads are covalently bound to antigens or antibodies as solid phases to bind antibodies or antigens in the liquid phase in a superior manner, thereby capturing and separating antigens and antibodies in the liquid phase. The immunochromatographic strip is qualitative detection method, this method can detect PCV2a strain, PCV2b strain, and PCV2d strain. The immunochromatographic strip had minimum detection limits of 102.89TCID50/0.1 mL, 103.19TCID50/0.1 mL, and 103.49TCID50/0.1 mL for PCV2a/LG, PCV2b/SH, and PCV2d/JH. The results of testing PEDV (CV777 strain), PRV (HB2000 strain), CSFV (WH-09 strain), PRRS (JXA1-R strain), PPV (WH-1 strain), and ASFV (SD strain) were negative. The agreement between the immunochromatographic strip and the ELISA kit was 93.33% (140/150) and the Kappa was 0.866 (Kappa > 0.81). On the premise of ensuring sensitivity, the most important feature of the immunochromatographic strip is that this method can save time when testing; results can be obtained within 5 to 10 min. Magnetic chemiluminescence immunoassay is quantitative detection method; this method can detect PCV2 Cap proteins in swine serum, the linear range of this method was 0.25 ng/mL to 32 ng/mL and R2 of the standard curve was 0.9993. The limit of detection (LOD) is 0.051 ng/mL and the limit of quantitation (LOQ) is 0.068 ng/mL. The agreement between the magnetic chemiluminescence immunoassay and the ELISA kit (test PCV2 Cap proteins) was 97.14% (68/70). This method took less than 30 min to achieve results, which is less than the ELISA kit. The results of this study show that immunochromatographic strip and magnetic chemiluminescence immunoassay for PCV2 antigens had great sensitivity and specificity, which lays the foundation for PCV2 clinical detection.

Background/Objectives: Porcine Circovirus 2 (PCV2) infection poses significant health and economic challenges to the global swine industry. The disease in pigs leads to lymphoid depletion, resulting in immunosuppression and increased susceptibility to co-infections with other bacterial and viral pathogens. This study evaluated the efficacy of two novel recombinant Newcastle disease virus (NDV) strain R2B vectored vaccines that express the cap gene of PCV2 alone and along with the transmembrane and cytoplasmic tail (TMCT) domains of the NDV F gene. The efficacy of the vaccine candidates was studied in mouse and pig models. Methods: Six-week-old BALB/c mice were divided into five groups and immunized intramuscularly three times at 14-day intervals with various vaccine candidates, namely rNDV-R2B-PCVcap-TMCT, rNDV-R2B-PCVcap, and CircoFLEX commercial vaccine, along with controls. Following immunization and PCV2d virus challenge, multiple assays assessed the immune responses in animal trials. In the pig animal trial, pigs were divided into four groups: a control group (PBS), NDV-vectored PCVcap-TMCT group, NDV-vectored-PCVcap group, and CircoFLEX vaccine group. Pigs were immunized intramuscularly twice at 28-day intervals. Blood samples were collected at regular intervals over 70 days to evaluate the humoral and cell-mediated immune responses. Results: Both mice and pigs’ trials indicated that the NDV-vectored PCV2 cap-TMCT vaccine candidate elicited superior immune responses. In mice, the rNDV-R2B-PCVcap-TMCT group showed enhanced humoral and cellular immunity, increased PCV2-specific antibody levels, higher CD4+/CD8+ ratio, elevated IFN-γ and TNF-α levels, decreased IL-10 levels, reduced viral loads, and minimal histopathological changes. In pigs, the NDV-vectored PCVcap-TMCT group demonstrated better antibody responses, cytokine profiles (IFN-γ and IL-10), and higher levels of PCV2-specific neutralizing antibodies against the PCV2a, PCV2b and PCV2d genotypes when compared to other groups. Conclusions: These findings suggest NDV-vectored PCVcap-TMCT vaccine candidate, expressing the cap gene of PCV2 along with the TMCT domain, offers a promising alternative for protecting against PCV2 infection, potentially addressing the challenges posed by emerging PCV2 strains in the swine industry.

The modernization of pig production has led to increasingly larger populations of pigs. This dynamic allows for accelerated production and ensures a steady pork supply but also facilitates the spread of infections. PCV2 is a ubiquitous virus and can cause PCV2-associated diseases, depending on production practices. This study aimed to evaluate the conditions of pig production in the state of Jalisco, Mexico, and correlate them with PCV2. A total of 4207 serum samples from 80 farms were analyzed. Epidemiological data were collected and used to investigate factors associated with PCV2 detection. A relative frequency of approximately 30% was detected, primarily in grower pigs maintained on multisite farms. Several production practices, particularly biosecurity measures, were associated with PCV2 on the analyzed farms.

Abstract Background Vaccination of pigs against PCV2 is usually performed around weaning when animals still have maternally derived antibodies (MDA). The present study aimed to assess the possible interference of MDA in the development of the PCV2-specific immune response after vaccination of commercial weaners. For this purpose, a PRRS-negative 600-sow farrow-to-finish farm was selected. Half of the sows were vaccinated and revaccinated with Porcilis® PCV ID against PCV2 7 and 3 weeks before farrowing. After farrowing, piglets were tested by AlphaLisa to select 72 animals with high and low levels of MDA. Groups were further subdivided and vaccinated intradermally with Porcilis® PCV ID at 21 or 28 days of age. Unvaccinated controls were also included. Animals were followed afterward for 42 days to examine the development of PCV2-specific antibodies and interferon-γ secreting cells (IFN-γ SC). Results The average titres of antibodies of the groups vaccinated in the presence of low or high MDA levels were similar at 28 and 42 days post-vaccination while in the controls the titres declined throughout the observation period. Results of vaccinating at 21 or 28 days of age were equivalent with regard to antibody development. Regarding the IFN-γ SC, vaccinated animals produced significant frequencies of IFN-γ SC by day 28. Again, no differences were observed between the groups with high or low antibody levels. Conclusion High levels of MDA did not interfere with the development of humoral and cell-mediated responses to Porcine circovirus 2 after intradermal vaccination at 21 or 28 days of age.

Abstract Background Porcine circovirus 2 (PCV-2) is one of the pathogens that leads to a growing and persistent threat in pigs. Thus, the development of serological detection methods for PCV-2 is of great necessity for clinical diagnosis as well as epidemiological investigations. This study aimed to establish an indirect enzyme-linked immunosorbent assay (ELISA) to examine antibodies against PCV-2 based on virus-like particles (VLPs). Results Recombinant PCV-2 Cap protein was expressed in the baculovirus-insect cells system and PCV-2 VLPs were observed over transmission electron microscopy (TEM). The PCV-2 VLPs were shown to have good immunogenicity in mice and stimulated a high level of PCV-2 antibody titers. Using PCV-2 VLPs as coating antigen, the indirect ELISA can detect PCV-2 antibodies in animals with diagnostic sensitivity and specificity of 98.33% and 93.33% compared to immunofluorescence assay (IFA), respectively. The intra- and inter-assay coefficient variations (CVs) were

Abstract Background Up to now, information on the levels of maternally-derived antibodies (MDA) against PCV-2 in suckling piglets born to sows vaccinated with different strategies is scarce in the literature. In the present observational study, the PCV-2-specific MDA titres from piglets from 109 farms (thirty 3-day-old and thirty 21-day-old piglets per farm) across four different European countries (France n = 30, Germany n = 27, Italy n = 22 and Spain n = 30) using different sow vaccination strategies (during gestation, as a gilt, as a piglet or never) were assessed. Results In all four countries, mean log PCV-2 MDA titres were higher in 3-day-old piglets than in the 3-week-old ones, being significant in most of all the comparisons performed. Within each country, the highest PCV-2-specific MDA titres were observed in the 3-day-old piglets born to sows vaccinated during gestation. Indeed, in the four countries, more than 60% of this subpopulation (3-day-old piglets from sows vaccinated during pregnancy) had the highest log PCV-2 titres detectable with the ELISA technique used in this study. The lowest MDA titres were more variable. Whereas in France and Germany the lowest titres corresponded to 21-day-old piglets born from sows vaccinated as a piglet, in Italy, they corresponded to 21-day-old piglets derived from sows vaccinated as a gilt and in Spain to 21-day-old piglets born from non-vaccinated sows. In this study, PCV-2-specific MDA titres at 3 and 21 days of age were not affected by sow parity. Conclusions Data obtained could be considered as a European global overview of PCV-2-specific MDA titres present in the pre-vaccinated piglet populations in different European countries, with titres tending to be higher in younger piglets, but with values variable among countries and sow vaccination strategies.

Porcine circovirus 2 (PCV2) has been recognized as a causative agent of porcine circovirus diseases (PCVDs) affecting the global swine industry. In this study, the genetic diversity of PCV2 strains circulating in Thailand between 2019 and 2020 was investigated using 742 swine clinical samples from 145 farms. The results showed PCV2-positive rates of 54.2% (402/742) and 81.4% (118/145) at the sample and farm levels, respectively. Genetic analysis of 51 Thai PCV2 genomic sequences showed that 84.3% (43/51) was PCV2d, 13.7% (7/51) was PCV2b and 1.9% (1/51) was PCV2b/2d recombinant virus. Surprisingly, the majority of the Thai PCV2d sequences from this study (69.77%, 30/43) formed a novel cluster on a phylogenetic tree and contained a unique 133HDAM136 on the ORF2 deduced amino acid sequence, which is in one of the previously identified immunoreactive domains strongly involved in virus neutralization. The PCV2b/2d recombinant virus also carried 133HDAM136. The emergence of the novel PCV2d strains predominating in Thailand was discussed. This study highlights the need for further investigations on the spreading of these PCV2d strains in other regions and the efficacy of current commercial vaccines.

Abstract Background Mycoplasma hyopneumoniae and Porcine circovirus type 2 are two economically important pathogens affecting growing pigs. Control and prevention of both diseases can be accomplished by vaccination, together with biosecurity and good management practices. Many commercial vaccines are available. The aim of this study was to assess the efficacy of Hyogen® and Circovac® administered mixed at weaning and to compare this protocol with a competitor ready-to-use (RTU) vaccine. Case presentation A randomised field trial was designed in a commercial farrow-to-finish farm located in France. A total of 641 pigs born from 54 different sows were included in this study. Piglets at weaning were allocated into three groups: the first one vaccinated with Hyogen® and Circovac® combined (group A), the second one vaccinated with a competitor RTU vaccine (group B) and the last one unvaccinated. Only minor local reactions for both vaccination groups could be observed which revealed a good safety of both protocols. Both vaccination schemes in this trial didn’t improve wean-to-slaughter growth performances but significantly reduced lung lesions, lung fissures and pleurisy at slaughter, produced a seroconversion for both M. hyopneumoniae and PCV-2 and significantly reduced the PCV-2 viral load in blood. When we compared groups A and B, we observed no significant differences in growth performances, mortality, clinical signs, percentages of affected lungs at slaughter, lung fissures and pleurisy, and no difference in pathogens detection. However, two statistical differences were observed between both vaccines: the mean lung lesion score and the percentage of extensive lung lesions were lower in group A. This is consistent with lower M. hyopneumoniae loads in the lower respiratory tract in pigs from group A but this difference was not statistically significant. Conclusions Results reported in this case study must be considered with caution since it was done in only one farm. In this trial, Hyogen® and Circovac® mixed together under field conditions offered a successful protection of growing pigs and significantly decreased the extension of lung lesions during a natural field challenge when compared with a competitor RTU vaccine.

Porcine circovirus 2 (PCV2) is one of the most important endemic swine pathogens, inducing immunosuppression in pigs and predisposing them to secondary bacterial or viral infections. Our previous studies show that PCV2 infection stimulated pig intestinal epithelial cells (IPEC-J2) to produce the secretory transforming growth factor-β (TGF-β), which, in turn, caused CD4+ T cells to differentiate into regulatory T cells (Tregs). This may be one of the key mechanisms by which PCV2 induces immunosuppression. Here, we attempt to identify the viral proteins that affect the TGF-β secretion, as well as the key amino acids that are primarily responsible for this occurrence. The three amino acids C35, S36 and V39 of the ORF4 protein are the key sites at which PCV2 induces a large amount of TGF-β production in IPEC-J2 and influences the frequency of Tregs. This may elucidate the regulatory effect of PCV2 on the Tregs differentiation from the perspective of virus structure and intestinal epithelial cell interaction, laying a theoretical foundation for improving the molecular mechanism of PCV2-induced intestinal mucosal immunosuppression in piglets.

The stillbirth, mummification, embryonic death, and infertility (SMEDI) syndrome is most commonly associated with porcine parvovirus 1 (PPV1) infections. Little is known about the occurrence of coinfections with SMEDI-associated pathogens and the associations among these pathogens. In our study, we included 40 SMEDI-affected litters from 18 different farms. In total, 158 out of 358 available fetuses from diagnostic transmittals were selected by systematic random sampling and examined for PCV2, PCV3, PPV1, and Leptospira spp. by q-PCR. Results from diagnostic materials showed the following results: in eleven farms, PCV2 was present; in nine farms, PPV1 was present; in five farms, PCV3 was present; and in two farms, Leptospira spp. was present. The detection of Leptospira spp. was significantly associated with a PCV2 coinfection (OR: 26.3; p < 0.001). PCV3 positivity resulted in a reduced probability of detecting PCV2 in the corresponding fetus (OR: 0.078; p = 0.008). Fetal maceration was associated with Leptospira spp. detection (OR: 8.6; p = 0.003), whereas mummification (p = 0.047), reduced crown-rump length (p < 0.001), and bodyweight (p = 0.001) of fetuses were significantly associated with PPV1 and PCV2 coinfection and thus, presumably, a shorter time to death after infection, indicating an enhanced negative effect on the development of fetuses with PCV2 + PPV1 coinfection.

Abstract Streptococcus suis is one of the most important bacterial swine pathogens affecting post-weaned piglets, causing mainly meningitis, arthritis and sudden death. It not only results in severe economic losses but also raises concerns over animal welfare and antimicrobial resistance and remains an important zoonotic agent in some countries. The definition and diagnosis of S. suis-associated diseases can be complex. Should S. suis be considered a primary or secondary pathogen? The situation is further complicated when referring to respiratory disease, since the pathogen has historically been considered as a secondary pathogen within the porcine respiratory disease complex (PRDC). Is S. suis a respiratory or strictly systemic pathogen? S. suis is a normal inhabitant of the upper respiratory tract, and the presence of potentially virulent strains alone does not guarantee the appearance of clinical signs. Within this unclear context, it has been largely proposed that co-infection with some viral and bacterial pathogens can significantly influence the severity of S. suis-associated diseases and may be the key to understanding how the infection behaves in the field. In this review, we critically addressed studies reporting an epidemiological link (mixed infections or presence of more than one pathogen at the same time), as well as in vitro and in vivo studies of co-infection of S. suis with other pathogens and discussed their limitations and possibilities for improvement and proposed recommendations for future studies.

Abstract Background Mycoplasma hyopneumoniae (Mhyo) and Porcine circovirus 2 (PCV-2) are two of the most significant infectious agents causing economic losses in the weaning to slaughter period. Due to their similar vaccination age, the objective of this study was to assess the efficacy of two already existing Mhyo (Hyogen®) and PCV-2 (Circovac®) vaccines when administered separately or combined (RTM) by means of Mhyo or PCV-2 experimental challenges. Results Seven groups of animals were included in the study, being three of them challenged with PCV-2, three with Mhyo and one composed of non-challenged, non-vaccinated pigs. Within each experimental challenge, non-vaccinated (NV) groups were compared with double vaccinated groups using the commercial products separated (VS) or combined (VC). Both vaccinated groups showed significant differences for most parameters measured regarding PCV-2 (serology, percentage of infected animals and viral load in tissues) and Mhyo (serology and gross lesions) when compared to NV groups. VS and VC offered similar results, being only significantly different the PCV-2 antibody values at different time points (higher in the VS group) of the study, although not at the termination day (21 days post-PCV-2 inoculation). Conclusion The present study expands the knowledge on the possibility of using two separate Mhyo and PCV-2 commercial vaccines as a RTM product, which offered equivalent virological, immunological and pathological outcomes as compared to these vaccines when used by separate.

African swine fever virus (ASFV), porcine circovirus 2 (PCV2), and pseudorabies virus (PRV) are important DNA viruses that cause reproductive disorders in sows, which result in huge losses in pig husbandry, especially in China. The multiplex qPCR assay could be utilized as a simultaneous diagnostic tool for field-based surveillance and the control of ASFV, PCV2, and PRV. Based on the conserved regions on the p72 gene of ASFV, the Cap gene of PCV2, the gE gene of PRV, and the porcine endogenous β-Actin gene, the appropriate primers and probes for a multiplex TaqMan real-time PCR test effective at concurrently detecting three DNA viruses were developed. The approach demonstrated high specificity and no cross-reactivity with major pathogens related to swine reproductive diseases. In addition, its sensitivity was great, with a detection limit of 101 copies/L of each pathogen, and its repeatability was excellent, with intra- and inter-group variability coefficients of

This study aimed to evaluate the efficacy of a new trivalent vaccine containing inactivated Porcine Circovirus 1-2a and 1-2b chimeras and a Mycoplasma hyopneumoniae bacterin administered to pigs around 3 weeks of age. This trivalent vaccine has already been proved as efficacious in a split-dose regimen but has not been tested in a single-dose scenario. For this purpose, a total of four studies including two pre-clinical and two clinical studies were performed. Globally, a significant reduction in PCV-2 viraemia and faecal excretion was detected in vaccinated pigs compared to non-vaccinated animals, as well as lower histopathological lymphoid lesion plus PCV-2 immunohistochemistry scorings, and incidence of PCV-2-subclinical infection. Moreover, in field trial B, a significant increase in body weight and in average daily weight gain were detected in vaccinated animals compared to the non-vaccinated ones. Circulation of PCV-2b in field trial A and PCV-2a plus PCV-2d in field trial B was confirmed by virus sequencing. Hence, the efficacy of this new trivalent vaccine against a natural PCV-2a, PCV-2b or PCV-2d challenge was demonstrated in terms of reduction of histopathological lymphoid lesions and PCV-2 detection in tissues, serum and faeces, as well as improvement of production parameters.

Porcine circovirus 2 (PCV2) causes immunosuppression. Piglets infected with PCV2 can develop enteritis. Given that the gut is the largest immune organ, however, the response of the gut’s immune system to PCV2 is still unclear. Here, IPEC-J2 cells with different treatments were co-cultured with PBMC or CD4+ T cells (Transwell). Flow cytometry and Western blotting revealed that PCV2-infected IPEC-J2 increased the frequency of CD4+ T cells among piglets’ peripheral blood mononuclear cells (PBMCs) and caused CD4+ T cells to undergo a transformation into Foxp3+ regulatory T cells (Treg cells) via activating CD4+ T ERK. Cytokines production and an inhibitor assay showed that the induction of Tregs by PCV2-infected IPEC-J2 was dependent on TGF-β induced by PCV2 in IPEC-J2, which was associated with the activation of NF-κB. Taken together, PCV2-infected IPEC-J2 activated NF-κB to stimulate the synthesis of TGF-β, which enhanced the differentiation of CD4+ T cells into Treg cells through the activation of ERK in CD4+ T cells. This information sheds light on PCV2′s function in the intestinal immune system and suggests a potential immunosuppressive mechanism for PCV2 infection.

Porcine circovirus 2 (PCV2) is the main pathogen of porcine circovirus-associated disease (PCVAD), which can cause considerable economic loss to the pig industry. The diagnosis of PCVAD is complicated and requires a series of clinical, pathological, and virological methods. Therefore, a rapid, highly sensitive, on-site, and visual diagnostic approach would facilitate dealing with the spread of PCV2. In this study, we intended to establish a new and effective PCV2 detection method through combining the no specific equipment requirement advantage of loop-mediated isothermal amplification (LAMP) with the property of clustered regular interspaced short palindromic repeats (CRISPR)/Cas12a system possessing the huLbCas12a collateral cleavage activity able to cleave single-stranded DNA fluorophore quencher probe sensor (designed as LAPM-CRISPR). Following a series of optimizations of its reaction conditions, this LAMP-CRISPR-based PCV2 detection could be conducted in constant temperature equipment, with the result reflected in a direct visual readout way. This established PCV2 detection approach presented fine sensitivity, rapidity, specificity, and reliability, as demonstrated by a low detectable limit of 1 copy/μL, completed within an hour, no cross-reaction with main porcine DNA or RNA viruses like PCV1, PCV3, and PEDV, and a 100% coincidence rate with that of the quantitative PCR (qPCR) method in the evaluation of 30 clinical blood samples, respectively. Therefore, this novel method makes rapid, on-site, visual, highly sensitive, and specific detection of PCV2 possible, facilitating the prevention of this pathogen in the field.

Four studies under preclinical and clinical conditions were performed to evaluate the efficacy of a new trivalent vaccine against Porcine circovirus 2 (PCV-2) infection. The product contained inactivated PCV-1/PCV-2a (cPCV-2a) and PCV-1/PCV-2b (cPCV-2b) chimeras, plus M. hyopneumoniae inactivated cell-free antigens, which was administered to piglets in a two-dose regime at 3 days of age and 3 weeks later. The overall results of preclinical and clinical studies show a significant reduction in PCV-2 viraemia and faecal excretion, and lower histopathological lymphoid lesions and PCV-2 immunohistochemistry scores in vaccinated pigs when compared to non-vaccinated ones. Furthermore, in field trial A, a statistically significant reduction in the incidence of PCV-2-subclinical infection, an increase in body weight from 16 weeks of age to slaughterhouse and an average daily weight gain over the whole period (from 3 days of age to slaughterhouse) was detected in the vaccinated group when compared to the non-vaccinated one. Circulation of PCV-2a in field trial A, and PCV-2b plus PCV-2d in field trial B was confirmed by virus sequencing. In conclusion, a double immunization with a cPCV-2a/cPCV-2b/M. hyopneumoniae vaccine was efficacious against PCV-2 infection by reducing the number of histopathological lymphoid lesions and PCV-2 detection in tissues, serum, and faeces, as well as reducing losses in productive parameters.

We report here high rates (47.5%, 48/101) of detection of porcine circovirus 2 (PCV2) in diarrheic pigs from three pig farms in the Dominican Republic. Seventeen of the PCV2 positive samples, representing the three pig farms, different age groups and sampling periods (2020–2021), were amplified for the complete PCV2 genome. Based on analysis of open reading frame 2 and complete genome sequences, the 17 PCV2 strains were assigned to the PCV2d genotype. Significant differences were observed in PCV2 detection rates between the vaccinated (20% (10/50)) and unvaccinated (62.5% (10/16) and 80% (28/35)) farms, corroborating previous observations that PCV2a-based vaccines confer protection against heterologous PCV2 genotypes. The present study is the first to report detection and molecular characterization of PCV2 from the Dominican Republic, warranting large-scale molecular epidemiological studies on PCV2 in pig farms and backyard systems across the country. For the first time, PCV2d was identified as the predominant PCV2 genotype in a study from the Caribbean region, suggesting that a genotype shift from PCV2b to PCV2d might be happening in the Caribbean region, which mirrored the current PCV2 genotype scenario in many other parts of the world. Besides PCV2, we also identified a pigeon circovirus-like virus, and a circular Replication-associated protein (Rep)-encoding single-stranded (CRESS) DNA virus, which was characterized for the complete genome. The CRESS DNA virus shared a similar genomic organization and was related to unclassified CRESSV2 DNA viruses (belonging to the Order Cirlivirales) from porcine feces in Hungary, indicating that related unclassified CRESS DNA viruses are circulating among pigs in different geographical regions, warranting further studies on the epidemiology and biology of these novel viruses.

The endoplasmic reticulum (ER) plays an essential role in Ca2+ concentration balance and protein biosynthesis. During infection, the virus needs to complete its life process with the help of ER. At the same time, ER also produces ER stress (ERS), which induces apoptosis to resist virus infection. Our study explored the Ca2+ concentration, ERS, and the apoptosis mechanism after porcine circovirus 2 (PCV2) infection. We show here that PCV2 infection induces the increased cytoplasmic Ca2+ level and PK-15 cell ER swelling. The colocalization of phospholipase C (PLC) and inositol 1,4,5-trisphosphate receptor (IP3R) in the cytoplasm was observed by laser confocal microscopy. Western blot and quantitative polymerase chain reaction experiments confirmed that PLC and IP3R expression levels increased after PCV2 infection, and Ca2+ concentration in the cytoplasm increased after virus infection. These results suggest that PCV2 infection triggers ERS of PK-15 cells via the PLC–IP3R–Ca2+ signaling pathway to promote the release of intracellular Ca2+ and led to cell apoptosis.

A whole virus, inactivated, Porcine Circovirus 2b (PCV2b) vaccine was submitted to a quantal assay of potency, as explained in detail in our companion paper [1]. To this purpose, twenty, 45-day old piglets, checked for maternally-derived antibody (MDA), were allocated to four groups of 5 animals each; these were vaccinated with 800/266/88/0 nanograms, respectively, of an inactivated PCV2b strain, consisting of two distinct virion populations. Twenty-six days later, all the pigs were challenged intranasally with the homologous PCV2b strain. In the presence of a clear dose-dependent protection in terms of viremia, no such effect was observed in terms of weight gain after challenge. The 800 and 266-ng payloads were associated with neutralizing antibody titers above the MDA levels in oral fluids. Higher levels of viremia in control and 88-ng groups [1] coincided with a higher Natural Killer activity of tracheobronchial lymph node cells from PCV2-infected pigs. The PCV2 ORF2-specific ELISPOT assay for IFN-g– secreting cells showed very few (2–4) ORF2-specific cells/105 peripheral blood mononuclear cells beyond the basal levels under our experimental conditions (non-significant differences among groups). Also, no significant differences were observed in the degree of lymphoid tissue hyperplasia among the different groups.

Porcine circovirus type 2 (PCV2) is the primary causative agent that causing porcine circovirus-associated disease (PCVAD). The open reading frame 5 (ORF5) protein is a newly discovered non-structural protein in PCV2, which the function in viral pathogenesis remains unknown. The aim of this study was to investigate the mechanism of PCV2 ORF5 protein on autophagy and viral replication. The pEGFP-tagged ORF5 gene was ectopic expressed in PK-15 cells and an ORF5-deficient PCV2 mutant strain (PCV2ΔORF5) were used to infected PK-15 cells. This study demonstrated that the ORF5 is essential for the of PCV2-induced autophagy. The ORF5 protein triggers the phosphorylation of PERK, eIF2α and the expression of downstream transcription factor ATF4. In addition, ORF5 protein activated the AMPK-ERK1/2-mTOR signaling pathways. These findings suggest that ORF5 play essential roles in the induction of autophagy by PCV2. We further revealed that PCV2 ORF5 promotes viral replication through PERK-eIF2α-ATF4 and AMPK-ERK1/2-mTOR pathways. In conclusion, we showed that PCV2 ORF5 induces autophagy to promote virus replication in PK-15 cells.

Porcine circovirus 2 (PCV2) has been recognized as an immunosuppressive pathogen. However, the crosstalk between this virus and its host cells in related signaling pathways remains poorly understood. In this study, the expression profiles of 84 genes involved in transforming growth factor-beta (TGF-β) signaling pathway were probed in PCV2b-infected primary porcine alveolar macrophages (PAMs) by using an RT2 profiler PCR array system. The protein expression levels of cytokines involved in the TGF-β signaling pathway were determined with a RayBiotech fluorescent Quantibody® porcine cytokine array system. Results showed that 48, 30, and 42 genes were differentially expressed at 1, 24, and 48 h after infection, respectively. A large number of genes analyzed by a co-expression network and implicated in transcriptional regulation and apoptosis were differentially expressed in PCV2b-infected PAMs. Among these genes, TGF-β, interleukin-10, CCAAT/enhancer-binding protein beta (C/EBPB), growth arrest, and DNA-damage-inducible 45 beta (GADD45B), and BCL2 were upregulated. By contrast, SMAD family member 1 (smad1) and smad3 were downregulated. These results suggested that the TGF-β signaling pathway was repressed in PAMs at the early onset of PCV2 infection. The inhibited apoptosis was indicated by the upregulated C/EBPB, GADD45B, and BCL2, and by the downregulated smad1 and smad3, which possibly increased the duration of PCV2 replication-permissive conditions and caused a persistent infection. Our study may provide insights into the underlying antiviral functional changes in the immune system of PCV2-infected pigs.

Porcine Circovirus 2 (PCV2) vaccines are based on either inactivated whole virion, or recombinant ORF2 capsid protein assembled into Virus-like Particles (VLPs). No data are available about the immunizing properties of free, non-assembled capsid protein. To investigate this issue, ORF2 of a reference PCV2b strain was expressed in a Baculovirus-based expression system without assembly into VLPs. The free purified protein was formulated into an oil vaccine at three distinct Ag payloads: 10.8/3.6/1.2 micrograms/dose. Each dose was injected intramuscularly into five, 37-day old piglets, carefully matched for maternally-derived antibody. Five control piglets were injected with sterile PBS in oil adjuvant. Twenty-eight days later, all the pigs were challenged intranasally with 105.3 TCID50 of PCV2b strain DV6503. After challenge infection, all the pigs remained in good clinical conditions. The recombinant vaccine did not induce significant antibody and PCV2-specific IFN-γ responses. ELISPOT and lymphocyte proliferation data confirmed poor induction of cell-mediated immunity. In terms of PCV2 viremia, there was no significant difference between vaccinated and control animals. The histological data indicated the absence of a detectable viral load and of PCVAD lesions in both vaccinated and control animals, as well as of histiocytes and multi-nucleated giant cells. We conclude that free, non-assembled ORF2 capsid protein does not induce protective immunity.

Vaccination against porcine circovirus 2 (PCV-2) is a common practice all over the world. Vaccines can prevent PCV-2-systemic disease (PCV-2-SD) outbreaks but not PCV-2 infection, which can be detectable in a percentage of vaccinated animals. Occasionally, PCV-2-SD is diagnosed in vaccinated farms. The objective of this study was to genotype the PCV-2 strains detected in vaccinated animals diagnosed with PCV-2-SD. Additionally, the evolution of the frequency of PCV-2 genotype detection at Spanish, European, and world levels was assessed. Fifty cases diagnosed as PCV-2-SD between 2009 and 2020 were included in this study. PCV-2 genotype was determined by sequencing the Cap gene region. Among them, only PCV-2b (23/50, 46%) and PCV-2d (27/50, 54%) genotypes were detected. Although the frequency of detection of these two genotypes was similar, their temporal distribution was different. Whereas most PCV-2b sequences (17/23, 74%) were detected between 2009 and 2012, PCV-2d sequences were obtained from 2013 to 2020. Indeed, a predominance of the PCV-2d genotype was observed from 2013 onwards, a trend also noticed at European and world levels. The results suggest that detection of particular genotypes in vaccinated animals probably reflects the general prevalence of the genotypes over time rather than genotype-specific vaccine-immunity escaping.

Porcine circovirus 2 (PCV2) is a major etiological agent for porcine circovirus-associated diseases and causes enormous economic losses in domestic and overseas swine production. However, there are currently no suitable cell models to study the cytopathic effects (CPE) of PCV2 in vitro, which severely restricts the study of PCV2 pathogenesis. In the present study, we established an immortalized porcine oral mucosal epithelial cell line (hTERT-POMEC) by introducing the hTERT gene into primary porcine oral mucosal epithelial cells (POMECs) derived from a neonatal, unsuckled piglet. The hTERT-POMEC cells have a homogeneous cobblestone-like morphology and retain the basic physiological properties of primary POMECs. No chromosome abnormality and tumorigenicity transformation was observed in immortalized hTERT-POMECs. Viral infection assays demonstrated that PCV2 propagated and caused CPE in hTERT-POMECs. We conclude that the immortalized cell line hTERT-POMEC is a crucial tool for further research into the pathogenesis of PCV2.

Porcine circovirus type 2 (PCV2) and Mycoplasma hyopneumoniae (Mhyo) are important swine pathogens for which vaccination is a key control strategy. Three separate studies were performed to evaluate the duration of immunity (DOI) conferred by a novel vaccine combining PCV2a/PCV2b and Mhyo into a ready-to-use formulation. In each study, three-week-old naïve piglets were vaccinated (Day 0) and challenged 23-weeks later (Day 159) with either PCV2a, PCV2b or Mhyo. Pigs were euthanized three-to-four-weeks post-challenge. Vaccinated pigs had significantly lower PCV2 viremia from Day 168 until Day 175 (PCV2a study) or until euthanasia (PCV2b study), respectively. Fecal shedding was significantly lower for PCV2a-challenged from Day 171 until Day 178, and for PCV2b-challenged from Day 172 until euthanasia. In the PCV2a challenge study, there were no differences among vaccinates and controls in terms of percent of pigs positive for PCV2 immunohistochemistry, histiocytic replacement, or lymphoid depletion. However, significant differences for immunohistochemistry and histiocytic replacement, not lymphoid depletion, were observed among vaccinates and controls following PCV2b challenge. Vaccination supposed a significant reduction in the mean percentage of Mhyo-like lesions in the lung. Percentages of lung tissues positive for Mhyo via immunohistochemistry were 49.3% and 67.1% for vaccinated and control groups, respectively. One dose of the novel PCV2a/PCV2b/Mhyo vaccine conferred robust protection against challenge 23-weeks later for all three fractions.

The presence of pseudorabies virus (PrV), porcine parvovirus (PPV) and porcine circovirus 2 (PCV2) was examined in sixty samples (spleen and lymph nodes) and thirty samples of sacral ganglia collected from non-vaccinated swine by virus isolation and polymerase chain reaction (PCR). Using PCR method PrV was detected in three samples, PPV in seven samples and six samples were found positive for PCV2. The phylogenetic analysis of the nucleotide sequences of three PrV isolates identified in this study showed high similarity and significant clustering within the PrV genotype I strains such as Kaplan and Bartha isolated from pigs in Hungary, strain Becker isolated in USA and strain Kolchis isolated in Greece. The nucleotide sequences of two PPV isolates showed high level of similarity with the strain Challenge isolated from pigs in UK, strain Kresse isolated in USA and strains 77 and LZ isolated in China. The phylogenetic analysis of the nucleotide sequences of two PCV2 isolates showed high level of similarity and significant clustering within genotype PCV2b strains such as NIVS-3, NIVS-5 and NIVS-6 isolated in Serbia, strain 3959 isolated in Austria, strain PM165 isolated from pigs in Brasil, and strain XT2008 isolated in China. The results of our study present the molecular characterization of PrV, PPV and PCV2 identified in swine in Republic of Montenegro. Besides that, these results confirmed that PCR is a very useful method for rapid detection of these viruses in subclinically infected swine.

The presence of porcine circovirus 2 and porcine parvovirus was examined in forty clinical samples of spleen, lymph nodes and lungs originating from non-vaccinated swine by polymerase chain reaction. All animals were reared in extensive livestock farming systems in different geographical districts of Republic of Srpska, Bosnia and Herzegovina. Porcine circovirus 2 DNA was detected in four lymph node and two spleen samples (15%), while porcine parvovirus DNA was identified in five lymph node samples (12.5%). The presence of both viruses was detected in three lymph node samples (7.5%). Partial nucleotide sequence of ORF1 gene of 2 porcine circovirus 2 and VP2 gene of 2 porcine parvovirus isolates was determined. The nucleotide sequences of two PCV2 isolates from RS-BIH included in phylogenetic typing are similar and cluster together with the strain Mantova isolated from domestic pigs in Italy, strains DE006-14 and DE222-13 isolated from pigs in Germany as well as with the strain Jvnan isolated from pigs in China. Also, analyzed PCV2 isolates were partially similar to the strain NIV-C SRB isolated from pigs in Serbia. The nucleotide sequences of two PPV isolates that were included in phylogenetic typing showed a high level of similarity with the strain Challenge isolated from pigs in UK, strain Kresse isolated from pigs in USA and strains 77 and LZ isolated from pigs in China.

Wild boar and domestic swine share several pathogens, including viruses responsible for reproductive failures, representing an important sanitary and economic risk for the swine industry. Among them, suid herpesvirus 1 (SuHV-1), porcine circovirus 2 (PCV2) and porcine parvovirus 1 (PPV1) are widely diffused in the wild boar population. Unfortunately, little is known about their pathogenetic mechanisms and impact on the reproductive parameters of wild animals. This study aims to investigate the presence of viruses responsible for reproductive failure in pregnant wild boar sows and their foetuses. The investigation was conducted on 46 pregnant wild boar and their foetuses by molecular analysis; a phylogenetic study was performed on the positive samples. All of the investigated pathogens were identified in sows, while only herpesvirus and circovirus were detected in the tissues of their foetuses. Phylogenetic analysis revealed that the viral sequences obtained from the positive wild boars were closely related to those previously identified in domestic swine belonging to the same study areas. The results suggest that SuHV-1 and PCV2 can infect wild boar foetuses, with a possible impact on wild boar reproductive performance. Moreover, our data highlight the importance of continuous monitoring of swine pathogens circulating in wild environments, so as to carry out adequate sanitary actions.

Mitochondrial dynamics is essential for the maintenance of cell homeostasis. Previous studies have shown that porcine circovirus 2 (PCV2) infection decreases the mitochondrial membrane potential and causes the elevation of reactive oxygen species (ROS), which may ultimately lead to mitochondrial apoptosis. However, whether PCV2 induce mitophagy remains unknown. Here we show that PCV2-induced mitophagy in PK-15 cells via Drp1 phosphorylation and PINK1/Parkin activation. PCV2 infection enhanced the phosphorylation of Drp1 and its subsequent translocation to mitochondria. PCV2-induced Drp1 phosphorylation could be suppressed by specific CDK1 inhibitor RO-3306, suggesting CDK1 as its possible upstream molecule. PCV2 infection increased the amount of ROS, up-regulated PINK1 expression, and stimulated recruitment of Parkin to mitochondria. N-acetyl-L-cysteine (NAC) markedly decreased PCV2-induced ROS, down-regulated Drp1 phosphorylation, and lessened PINK1 expression and mitochondrial accumulation of Parkin. Inhibition of Drp1 by mitochondrial division inhibitor-1 Mdivi-1 or RNA silencing not only resulted in the reduction of ROS and PINK1, improved mitochondrial mass and mitochondrial membrane potential, and decreased mitochondrial translocation of Parkin, but also led to reduced apoptotic responses. Together, our study shows that ROS induction due to PCV2 infection is responsible for the activation of Drp1 and the subsequent mitophagic and mitochondrial apoptotic responses.

Porcine circovirus type 2 (PCV2) induces autophagy via the 5′ adenosine monophosphate-activated protein kinase (AMPK)/extracellular signal-regulated kinase (ERK)/tuberous sclerosis complex 2 (TSC2)/mammalian target of rapamycin (mTOR) pathway in pig kidney PK-15 cells. However, the underlying mechanisms of AMPK activation in autophagy induction remain unknown. With specific inhibitors and RNA interference (RNAi), we show that PCV2 infection upregulated calcium/calmodulin-dependent protein kinase kinase-beta (CaMKKβ) by increasing cytosolic Ca2+ via inositol 1,4,5-trisphosphate receptor (IP3R). Elevation of cytosolic calcium ion (Ca2+) did not seem to involve inositol 1,4,5-trisphosphate (IP3) release from phosphatidylinositol 4,5-bisphosphate (PIP2) by phosphoinositide phospholipase C-gamma (PLC-γ). CaMKKβ then activated both AMPK and calcium/calmodulin-dependent protein kinase I (CaMKI). PCV2 employed CaMKI and Trp-Asp (WD) repeat domain phosphoinositide-interacting protein 1 (WIPI1) as another pathway additional to AMPK signaling in autophagy initiation. Our findings could help better understanding of the signaling pathways of autophagy induction as part of PCV2 pathogenesis. Further research is warranted to study if PCV2 interacts directly with IP3R or indirectly with the molecules that antagonize IP3R activity responsible for increased cytosolic Ca2+ both in PK-15 cells and PCV2-targeted primary cells from pigs.

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.