Can immunocrit be used as a monitoring tool for swine vaccination and infection studies?

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 < 0.05) S/P values (S/P ratio = 0.74) than NV (S/P ratio = 0.39) pigs only after challenge at SD42. On the other hand, serum total protein obtained by refractometer (STPr) were maintained from SD0 to SD21 and increased in both groups from SD21 to SD42. Correlations between techniques were low to moderate, being the most robust ones found between immunocrit and optical refractometry (ρ = 0.41) and immunocrit with γ-globulins (ρ = 0.39). In a subset of sera, the proteinogram technique was applied to the whole serum and the supernatant of the immunocrit, with the objective to characterize indirectly the immunocrit fraction. The latter one included all protein types detectable through the proteinogram, with percentages varying between 64.3% (γ-globulins) and 82% (ß-globulins). CONCLUSION: The immunocrit technique represented a fraction of the total serum proteins, with low to moderate correlation with all the complementary techniques measured in this study. Its determination at different time points did not allow monitoring the effect of vaccination and/or infection using PCV-2 as a pathogen model.

[An in vitro self-ligation-based method for constructing infectious DNA clone of porcine circovirus type 2].

The aim of this study was to establish a rapid method for constructing infectious clones of porcine circovirus type 2 (PCV2). In this study, we constructed circular infectious clones of PCV2 by seamless cloning technology, using the clinically isolated strain PCV2-LX as a template. Meanwhile, this method was compared with the conventional restriction-ligation approach, focusing on the in vitro circularization (self-ligation) process of the genome and the growth characteristics of rescued viruses. The results showed that this method eliminates the need to analyze and introduce restriction endonuclease sites, thus avoiding the complexities associated with traditional restriction enzyme-based cloning steps. It offers a simple and rapid operation, enabling more efficient editing of the PCV2 genome. The infectious clones constructed using this method could be successfully rescued through liposome transfection, resulting in the production of recombinant viruses that could be stably passaged. Moreover, the recombinant viruses rescued by this method exhibited enhanced proliferative capacity in PK-15 cells and 3D4/31 cells (immortalized porcine alveolar macrophages). In conclusion, this study has established a novel reverse genetics system for PCV2, providing a new strategy for the development of PCV2 genetic engineering vaccines. Additionally, it serves as a reference for the construction of infectious clones for other emerging circoviruses such as PCV3 and PCV4.

Frequency of PCV-2 viremia in nursery piglets from a Spanish swine integration system in 2020 and 2022 considering PRRSV infection status.

BACKGROUND: Porcine circovirus 2 (PCV-2) poses a significant economic threat for the swine industry, causing a range of diseases collectively referred to as porcine circovirus diseases (PCVDs). Despite PCV-2 vaccine effectiveness, the need for monitoring infectious pressure remains. PCV-2 coinfection with other pathogens like porcine reproductive and respiratory syndrome virus (PRRSV) can exacerbate disease severity and lead to PCV-2-systemic disease cases. Monitoring both PRRSV and PCV-2 in co-infected farms is crucial for an effective management and vaccination programs. The present cross-sectional study aimed to determine PCV-2 antibody levels in piglets at weaning and PCV-2 and PRRSV viremia in pooled serum samples at weaning (vaccination age) and at 6 and 9 weeks of age from a Spanish swine integration system in 2020 (48 farms) and in 2022 (28 out of the 48 analysed previously). RESULTS: The frequency of PCV-2 detection in pools of piglet sera was 2.1% (2020) and 7.1% (2022) at vaccination age but increased at the end of the nursery period (10.4% in 2020 and 39.3% in 2022) in both years. Co-infections between PCV-2 and PRRSV were detected in a significant proportion of PRRSV positive farms (15% in 2020, and 60% in 2022). PCV-2 antibody levels (ELISA S/P ratios) at weaning were lower in PCV-2 qPCR positive farms at different sampling time-points (0.361 in 2020 and 0.378 in 2022) compared to PCV-2 qPCR negative ones (0.587 in 2020 and 0.541 in 2022). The 28 farms tested both years were classified in four different epidemiological scenarios depending on their PCV-2 virological status. Those PCV-2 qPCR negative farms in 2020 that turned to be positive in 2022 had a statistically significant increase of PRRSV RT-qPCR detection and a PCV-2 antibody levels reduction, facts that were not observed in the rest of the scenarios. CONCLUSION: This epidemiological study in farms from the same integration system determined the occurrence, in 2020 and in 2022, of PCV-2 and PRRSV infections in piglets during the nursery period by using pooled serum samples.

A multiplex real-time RT-PCR system to simultaneously diagnose 16 pathogens associated with swine respiratory disease.

AIMS: Swine respiratory disease (SRD) is a major disease complex in pigs that causes severe economic losses. SRD is associated with several intrinsic and extrinsic factors such as host health status, viruses, bacteria, and environmental factors. Particularly, it is known that many pathogens are associated with SRD to date, but most of the test to detect those pathogens can be normally investigated only one pathogen while taking time and labor. Therefore, it is desirable to develop rapidly and efficiently detectable methods those pathogens to minimize the damage caused by SRD. METHODS AND RESULTS: We designed a multiplex real-time RT-PCR (RT-qPCR) system to diagnose simultaneously 16 pathogens, including nine viruses and seven bacteria associated with SRD, on the basis of single qPCR and RT-qPCR assays reported in previous studies. Multiplex RT-qPCR system we designed had the same ability to single RT-qPCR without significant differences in detection sensitivity for all target pathogens at minimum to maximum genomic levels. Moreover, the primers and probes used in this system had highly specificity because the sets had not been detected pathogens other than the target and its taxonomically related pathogens. Furthermore, our data demonstrated that this system would be useful to detect a causative pathogen in the diagnosis using oral fluid from healthy pigs and lung tissue from pigs with respiratory disorders collected in the field. CONCLUSIONS: The rapid detection of infected animals from the herd using our system will contribute to infection control and prompt treatment in the field.

High levels of maternally derived antibodies do not significantly interfere with the development of humoral and cell-mediated responses to Porcine circovirus 2 after intradermal vaccination.

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.

[Construction of a replicative expression vector based on the porcine circovirus 2 replicon].

The antigen gene expression level of a DNA vaccine is the key factor influencing the efficacy of the DNA vaccine. Accordingly, one of the ways to improve the antigen gene expression level of a DNA vaccine is to utilize a plasmid vector that is replicable in eukaryotic cells. A replicative DNA vaccine vector pCMVori was constructed based on the non-replicative pcDNA3.1 and the replicon of porcine circovirus 2 (PCV2) in this study. An EGFP gene was cloned into pCMVori and the control plasmid pcDNA3.1. The two recombinant vectors were transfected into PK-15 cell, and the plasmid DNA and RNA were extracted from the transfected cells. Real-time PCR was used to determine the plasmid replication efficiency of the two plasmids using plasmid before and after Bcl Ⅰ digestion as templates, and the transcription level of the Rep gene in PCV2 replicon was detected by RT-PCR. The average fluorescence intensity of cells transfected with the two plasmids was analyzed with software Image J, and the transcription level of EGFP was determined by means of real-time RT-PCR. The results showed that the replication efficiency of pCMVori in PK-15 cells incubated for 48 h was 136%, and the transcriptions of Rep and Rep' were verified by RT-PCR. The average fluorescence intensity of the cells transfected with pCMVori-EGFP was 39.14% higher than that of pcDNA3.1-EGFP, and the transcription level of EGFP in the former was also 40% higher than that in the latter. In conclusion, the DNA vaccine vector pCMVori constructed in this study can independently replicate in eukaryotic cells. As a result, the expression level of cloned target gene was elevated, providing a basis for developing the pCMVori-based DNA vaccine.

Porcine Circovirus 2 Increases the Frequency of Transforming Growth Factor-ß via the C35, S36 and V39 Amino Acids of the ORF4.

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-ß (TGF-ß), 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-ß 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-ß 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.

Molecular characterization and phylogenetic analysis of porcine circovirus 2 from Kerala, India.

Porcine circovirus type 2 (PCV2), the causative agent of porcine circovirus-associated diseases (PCVADs), has a worldwide distribution, and is considered as one of the most important emerging viral pathogens of economic importance. In Kerala, a total of 62 tissue samples were collected during post mortem from pigs suspected to have died of PCV2 infection. The animals exhibited symptoms like respiratory illness, gradual wasting, rough hair coat, polypnoea, dyspnoea, pallor, diarrhoea, icterus, etc. PCV2 was detected in 36 (58.06%) samples by PCR. Phylogenetic analyses of complete ORF2, and complete genome sequences were carried out and genotypes 2d, 2 h and 2b were detected. The genotype predominant in Kerala was 2d. It was observed that genotypes 2 h and 2b have been recently introduced into North Kerala as it was not detected in the region prior to 2016. Close relationship of Kerala sequences with sequences from Tamil Nadu, Uttar Pradesh and Mizoram were noticed in the phylogenetic tree and also at the amino acid level. A unique K243N mutation was observed in one of the samples. It was also noticed that the most variable amino acid position in ORF2 was 169 where the occurrence of three possible amino acids were observed. The results of the study indicate that multiple genotypes of PCV2 are prevalent in pigs in Kerala and that the percent positivity is higher than that recorded in the State previously. Supplementary Information: The online version contains supplementary material available at 10.1007/s13337-023-00814-1.

Emergence of novel porcine circovirus 2d strains in Thailand, 2019-2020.

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.

Examination on the Occurrence of Coinfections in Diagnostic Transmittals in Cases of Stillbirth, Mummification, Embryonic Death, and Infertility (SMEDI) Syndrome in Germany.

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.

Production of virus-like particles of porcine circovirus 2 in baculovirus expression system and its application for antibody detection.

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 < 10% in a batch, and < 15% in different batches, indicating good repeatability. There was no cross-reaction of this ELISA with antibodies against other porcine viruses. A total of 170 serum samples collected from different pig farms in China were tested for PCV-2 antibodies, and 151 (88.8%) samples were PCV-2 antibody positive. CONCLUSION: Our findings suggest that this ELISA was rapid, specific, and reproducible and can be used for large-scale serological investigations of PCV-2 antibodies in pigs.

Molecular characterization of porcine circovirus-2 and -3 in pigs in Tanzania.

Like in many other African countries, pig production is increasing in Tanzania. To support farmers and stakeholders, it is important to increase our understanding of porcine pathogens present in the country. Currently, little is known about the circulation of porcine circovirus-2 (PCV-2) and porcine circovirus-3 (PCV-3). For this reason, samples from 124 pigs collected throughout the country between 2018 to 2022 were screened by PCR for the presence of PCV-2 and PCV-3. Sequencing and phylogenetic analysis of positive amplicons identified two PCV-2 genotypes (a and d). Limited genetic heterogenicity was observed among the PCV-3. This study provides important data on pathogens present in pigs in Tanzania and should be of use veterinary authorities involved in porcine disease management.

Development of a TaqMan-Probe-Based Multiplex Real-Time PCR for the Simultaneous Detection of African Swine Fever Virus, Porcine Circovirus 2, and Pseudorabies Virus in East China from 2020 to 2022.

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 ß-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 <2%. Applying this assay to detect 383 field specimens collected from 2020 to 2022, the survey data displayed that the ASFV, PCV2, and PRV single infection rates were 22.45%, 28.46%, and 2.87%, respectively. The mixed infection rates of ASFV + PCV2, ASFV + PRV, PCV2 + PRV, and ASFV + PCV2 + PRV were 5.22%, 0.26%, 1.83%, and 0.26%, respectively. Overall, the assay established in this study provides an effective tool for quickly distinguishing the viruses causing sow reproductive disorders, suggesting its huge clinical application value in the diagnosis of swine diseases.

Porcine circoviruses in wild boars in Nagano Prefecture, Japan.

We investigated the prevalence of porcine circovirus 2 (PCV2) and 3 (PCV3) in wild boars captured or found dead in Nagano Prefecture in 2020. Based on PCR testing, 21 of 254 (8.3%) wild boars were PCV2-positive and 43 of 256 (16.8%) wild boars were PCV3-positive, 5 of 253 (2.0%) wild boars were both PCV2-positive and PCV3-positive. The frequency of detecting PCV3 in wild boars was significantly higher in adults than in juveniles (P=0.014). The PCV2-positive wild boars were found in all districts except for the North Alps and Hokushin, while PCV3-positive wild boars were found in all districts except for Suwa. This is the first report of PCV2 and PCV3 detected in wild boars in Japan.

Efficacy Studies against PCV-2 of a New Trivalent Vaccine including PCV-2a and PCV-2b Genotypes and Mycoplasma hyopneumoniae When Administered at 3 Weeks of Age.

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.

TGF-ß from the Porcine Intestinal Cell Line IPEC-J2 Induced by Porcine Circovirus 2 Increases the Frequency of Treg Cells via the Activation of ERK (in CD4+ T Cells) and NF-κB (in IPEC-J2).

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-ß 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-ß, 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 Activates the PERK-Reactive Oxygen Species Axis To Induce p53 Phosphorylation with Subsequent Cell Cycle Arrest at S Phase in Favor of Its Replication.

Porcine circovirus type 2 (PCV2), the causative agent of porcine circovirus-associated diseases (PCVAD), is known to induce oxidative stress, activate p53 with induction of cell cycle arrest, and trigger the PERK (protein kinase R-like endoplasmic reticulum kinase) branch of the endoplasmic reticulum (ER) stress pathway. All these cellular responses could enhance PCV2 replication. However, it remains unknown whether PERK activation by PCV2 is involved in p53 signaling with subsequent changes of cell cycle. Here, we demonstrate that PCV2 infection induced cell cycle arrest at S phase to favor its replication via the PERK-reactive oxygen species (ROS)-p53 nexus. PCV2 infection promoted phosphorylation of p53 (p-p53) at Ser15 in porcine alveolar macrophages. Inhibition of PERK by RNA silencing downregulated total p53 (t-p53) and p-p53. Treatment with the MDM2 inhibitor nutlin-3 led to partial recovery of t-p53 in perk-silenced and PCV2-infected cells. perk silencing markedly downregulated ROS production. Scavenging of ROS with N-acetylcysteine (NAC) of PCV2-infected cells downregulated t-p53 and p-p53. Increased accumulation of p-p53 in the nuclei during PCV2 infection could be downregulated by silencing of perk or NAC treatment. Further studies showed that perk silencing or NAC treatment alleviated S phase accumulation and downregulated cyclins E1 and A2 in PCV2-infected cells. These findings indicate that the PCV2-activated PERK-ROS axis promotes p-p53 and contributes to cell cycle accumulation at S phase when more cellular enzymes are available to favor viral DNA synthesis. Overall, our study provides a novel insight into the mechanism how PCV2 manipulates the host PERK-ROS-p53 signaling nexus to benefit its own replication via cell cycle arrest. IMPORTANCE Coinfections or noninfectious triggers have long been considered to potentiate PCV2 infection, leading to manifestation of PCVAD. The triggering mechanisms remain largely unknown. Recent studies have revealed that PERK-mediated ER stress, oxidative stress, and cell cycle arrest during PCV2 infection are conducive to viral replication. However, how PCV2 employs such host cell responses requires further research. Here, we provide a novel mechanism of PCV2-induced ER stress and enhanced viral replication: the PCV2-activated PERK-ROS-p53 nexus increases S phase cell population, a cell cycle period of DNA synthesis favorable for PCV2 replication. The fact that PCV2 deploys the simple ROS molecules to activate p53 to benefit its replication provides novel insights into the triggering factors, that is, certain stimuli or management measures that induce ER stress with subsequent generation of ROS would exacerbate PCVAD. Use of antioxidants is justified on farms where PCVAD is severe.

Descriptive analyses of maternally-derived antibody levels against porcine circovirus 2 (PCV-2) in 3- and 21-day-old piglets from farms of four European countries using different vaccination protocols in sows.

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.

Histopathological findings of the nasopharynx-associated lymphoid tissue of pigs co-infected with porcine circovirus 2 and porcine reproductive and respiratory syndrome virus.

Porcine circovirus 2 (PCV2) causes porcine circovirus-associated disease, and co-infection with porcine reproductive and respiratory syndrome virus (PRRSV) severely affects the pig breeding industry. Both viruses target the macrophages in lymphoid tissues. Various porcine pathogens enter via the nasal cavity, and the nasopharynx-associated lymphoid tissue (NALT) acts as the mucosal immune system. However, the pathological analysis has not progressed. This study aimed to histologically examine the NALT of pigs with suspected PCV2 and PRRSV infections. Six pigs were subjected to necropsy, and their NALT, tonsils, and mesenteric lymph nodes were collected. Macrophages, lymphocytic depletion, multinucleated giant cells, intracytoplasmic inclusion bodies, and neutrophil infiltration increased in the NALT. In situ hybridization revealed positive signals for PCV2 in the NALT of all pigs and PRRSV in the NALT of three pigs. PCV2-positive macrophages were mainly identified in the follicles, whereas PRRSV-positive tissues were found primarily around the crypt and directly below the epithelium. Quantitative PCR revealed 108-1010 copies of PCV2 DNA/µL and 102-104 copies of PRRSV DNA/µL in the NALT. Therefore, both PCV2 and PRRSV were detected in the NALT of pigs. In conclusion, the infection and replication of both viruses in the NALT and tonsils may suppress host immunity and promote co-infection with other pathogens.

LAMP Coupled CRISPR-Cas12a Module for Rapid, Sensitive and Visual Detection of Porcine Circovirus 2.

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.