A multiplex digital PCR assay for detection and quantitation of porcine circovirus type 2 and type 3.

Porcine circovirus (PCV) has become a major pathogen, causing major economic losses in the global pig industry, and PCV type 2 (PCV2) and 3 (PCV3) are distributed worldwide. We designed specific primer and probe sequences targeting PCV2 Cap and PCV3 Rap and developed a multiplex crystal digital PCR (cdPCR) method after optimizing the primer concentration, probe concentration, and annealing temperature. The multiplex cdPCR assay permits precise and differential detection of PCV2 and PCV3, with a limit of detection of 1.39 × 101 and 1.27 × 101 copies/reaction, respectively, and no cross-reaction with other porcine viruses was observed. The intra-assay and interassay coefficients of variation (CVs) were less than 8.75%, indicating good repeatability and reproducibility. To evaluate the practical value of this assay, 40 tissue samples and 70 feed samples were tested for both PCV2 and PCV3 by cdPCR and quantitative PCR (qPCR). Using multiplex cdPCR, the rates of PCV2 infection, PCV3 infection, and coinfection were 28.45%, 1.72%, and 12.93%, respectively, and using multiplex qPCR, they were 25.00%, 0.86%, and 4.31%, respectively This highly specific and sensitive multiplex cdPCR thus allows accurate simultaneous detection of PCV2 and PCV3, and it is particularly well suited for applications that require the detection of small amounts of input nucleic acid or samples with intensive processing and complex matrices.

Implementation of point-of-care platforms for rapid detection of porcine circovirus type 2.

BACKGROUND: Porcine circovirus type 2 (PCV2) infection is ubiquitous around the world. Diagnosis of the porcine circovirus-associated disease requires clinic-pathological elements together with the quantification of viral loads. Furthermore, given pig farms in regions lacking access to sufficient laboratory equipment, developing diagnostic devices with high accuracy, accessibility, and affordability is a necessity. OBJECTIVES: This study aims to investigate two newly developed diagnostic tools that may satisfy these criteria. METHODS: We collected 250 specimens, including 170 PCV2-positive and 80 PCV2-negative samples. The standard diagnosis and cycle threshold (Ct) values were determined by quantitative polymerase chain reaction (qPCR). Then, two point-of-care (POC) diagnostic platforms, convective polymerase chain reaction (cPCR, qualitative assay: positive or negative results are shown) and EZtargex (quantitative assay: Ct values are shown), were examined and analyzed. RESULTS: The sensitivity and specificity of cPCR were 88.23% and 100%, respectively; the sensitivity and specificity of EZtargex were 87.65% and 100%, respectively. These assays also showed excellent concordance compared with the qPCR assay (κ = 0.828 for cPCR and κ = 0.820 for EZtargex). The statistical analysis showed a great diagnostic power of the EZtargex assay to discriminate between samples with different levels of positivity. CONCLUSIONS: The two point-of-care diagnostic platforms are accurate, rapid, convenient and require little training for PCV2 diagnosis. These POC platforms can discriminate viral loads to predict the clinical status of the animals. The current study provided evidence that these diagnostics were applicable with high sensitivity and specificity in the diagnosis of PCV2 infection in the field.

Pluck-pools as diagnostic samples for detecting porcine reproductive and respiratory syndrome virus and porcine circovirus type 2 in porcine abortion material and stillbirths.

Investigating infectious agents in porcine abortion material and stillborn piglets poses challenges for practitioners and diagnostic laboratories. In this study, pooled samples of individual reference organs (thymus and heart) from a total of 1000 aborted fetuses and stillborn piglets were investigated using quantitative PCR protocols for porcine reproductive and respiratory syndrome virus 1 (PRRSV-1) and porcine circovirus type 2 (PCV2). Simultaneously, a pluck-pool containing equivalent portions of fetal thymus, heart, and lung tissue was collected, frozen at - 20 °C, and re-analyzed when a certain amount of either PRRSV-1 RNA or PCV2 DNA was detected in individual reference organs. Thirteen pluck-pools were assessed for PRRSV-1, all being PCR-positive. For PCV2, 11 of 15 pluck-pools investigated were PCR-positive. In all pluck-pools testing negative, viral loads in individual pools were low. This study indicates that pluck-pools can be valuable diagnostic material and the consolidation of multiple organs through a single RNA/DNA extraction optimizes the utilization of available laboratory resources. Additional research is required to assess the feasibility of follow-up investigations and to accurately define criteria for interpretation of viral loads in a clinical context.

Simultaneous detection and differentiation of DuCV-1 and DuCV-2 by high-resolution melting analysis.

Birds infected with duck circovirus (DuCV) can potentially cause immunosuppression by damaging lymphoid tissues, causing great losses in the duck breeding industry. Duck circovirus can be divided into two genotypes (DuCV-1 and DuCV-2), but simultaneous detection and differentiation of DuCV-1 and DuCV-2 by high-resolution melting (HRM) analysis is still lacking. Here, we designed specific primers according to the sequence characteristics of the newly identified ORF3 gene and then established a PCR-HRM method for the simultaneous detection and differentiation of DuCV-1 and DuCV-2 via high-resolution melting analysis. Our data showed that the established PCR-HRM assay had the advantages of specificity, with the lowest detection limits of 61.9 copies/µL (for DuCV-1) and 60.6 copies/µL (for DuCV-2). The melting curve of the PCR-HRM results indicated that the amplification product was specific, with no cross-reaction with common waterfowl origin pathogens and a low coefficient of variation less than 1.50% in both intra-batch and inter-batch repetitions, indicating the advantages of repeatability. We found that the percentage of DuCV-2-positive ducks was higher than that of DuCV-1-positive ducks, with 8.62% rate of DuCV-1 and DuCV-2 coinfection. In addition, we found DuCV-2-positive in geese firstly. In conclusion, this study provides a candidate PCR-HRM assay for the detection and accurate differentiation of DuCV-1 and DuCV-2 infection, which will help us for further epidemiological surveillance of DuCVs.

Development of recombinant capsid protein-based single serum dilution ELISA for sero-detection of porcine circovirus type 2 infection in pigs.

Porcine Circovirus 2 (PCV2) is the major causative agent of postweaning multisystemic wasting syndrome (PMWS) of swine and is one of the reasons for severe economic loss in swine industry. In India, there is a considerable prevalence rate of PCV2 infection in pig population, PCV2d being the most prominent genotype. Proper sero-diagnosis and sero-surveillance of the disease is formulated as an effective control measure. In this study, a recombinant capsid protein-based single serum dilution indirect ELISA was developed for determination of antibody titre of the infected pigs. The capsid protein (Cap) of PCV2d was produced in Saccharomyces cerevisiae cells and the capsid protein was purified by affinity chromatography. This recombinant protein was used as a coating antigen to develop a cost effective, highly sensitive and specific single serum dilution ELISA. The in-house developed ELISA was optimized to be used in a 1:200 single serum dilution. The developed ELISA along with a commercial ELISA kit were compared with a sensitive immuno-peroxidase assay (IPMA) by receiver-operating characteristics (ROC) test. Our results showed that the developed single serum dilution ELISA had a higher sensitivity and specificity in comparison to the commercial ELISA. The area under the ROC curve (AUC) also confirmed that the developed ELISA had a better overall diagnostic performance in comparison to the commercial ELISA kit.

Phylogenetic Analysis and Serological Investigation of Porcine Circovirus Indicates Frequent Infection with Various Subtypes.

Porcine circoviruses (PCVs) are notorious for triggering severe diseases in pigs and causing serious economic losses to the swine industry. In the present study, we undertook a comprehensive approach for the investigation of PCV prevalence, including the phylogenetic analysis of obtained PCV sequences, the determination of major circulating genotypes and serological screening based on different recombinant Cap proteins with specific immunoreactivity. Epidemiological surveillance data indicate that PCV2d and PCV3a are widely distributed in Southwest China, while PCV4 has only sporadic circulation. Meanwhile, serological investigations showed high PCV2 antibody positivity in collected serum samples (>50%), followed by PCV4 (nearly 50%) and PCV3 (30-35%). The analysis supports different circulation patterns of PCV2, PCV3 and PCV4 and illustrates the PCV2/PCV3 genetic evolution characteristics on a nationwide basis. Taken together, our findings add up to the current understanding of PCV epidemiology and provide new tools and insight for PCV antiviral intervention.

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.

Development of a Gold Nanoparticle-Based Immunochromatographic Strip for Rapid Detection of Porcine Circovirus Type 2.

Porcine circovirus type 2 (PCV2) is an important swine infectious pathogen that seriously threatens the global swine industry. PCV2 Cap protein is the only structural and the main immunogenic protein constituting the viral capsid. In this study, a gold nanoparticle-based immunochromatographic strip with high sensitivity and specificity was developed which could be used for rapid detection of PCV2 virions or Cap protein in research. The visual detection limit of the strip was 103.18 50% tissue culture infective does (TCID50)/mL for PCV2, and 2.03 µg/mL for PCV2 Cap protein. No cross-reactivity was observed with the PCV1 and PCV3 Cap proteins and other common swine pathogens such as porcine reproductive and respiratory syndrome virus, classical swine fever virus, pseudorabies virus, porcine epidemic diarrhea virus, porcine parvovirus, and swine influenza virus. The repeatability of the strip was good. The stability of the strip was perfect for 12 months in a dry state at room temperature. Visual results could be obtained within 5 min by simply inserting the strip into the diluted sample. The strip is a time-saving, labor-saving, and reliable tool for testing of PCV2 virions or Cap protein in research. The idea of this study might open a new perspective for the application of the strip. IMPORTANCE Porcine circovirus type 2 (PCV2) Cap protein is the only structural and the main immunogenic protein constituting the viral capsid. Although many methods can be used to identify PCV2 or PCV2 Cap protein in vaccine research, they usually require high workload and time. The developed strip can specifically detect PCV2 virions or Cap protein, and visual qualitative results can be obtained within 5 min by simply diluting the sample and inserting the strip into the sample. The final value of the strip is providing a simple and time-saving method for real-time monitoring of PCV2 antigen in vaccine research with reliable results, such as the different stages of PCV2 Cap protein expression and purification, as well as the different stages of PCV2 reproduction and purification.

Unknown Circovirus in Immunosuppressed Patient with Hepatitis, France, 2022.

Hepatitis of undetermined origin can be caused by a wide variety of pathogens, sometimes emerging pathogens. We report the discovery, by means of routine shotgun metagenomics, of a new virus belonging to the family Circoviridae, genus Circovirus, in a patient in France who had acute hepatitis of unknown origin.

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.

Development of multiplex PCR assay for simultaneous detection of African swine fever, porcine circo and porcine parvo viral infection from clinical samples.

A diagnostic method for simultaneously detecting and distinguishing African Swine Fever (ASF), porcine circovirus type 2 (PCV2), and porcine parvovirus (PPV) in clinical specimens is critical for differential diagnosis, monitoring, and control in the field. Three primer pairs were designed and used to create a multiplex PCR assay. In addition, 356 porcine post mortem tissue samples from various parts of India's North Eastern region were tested by the developed multiplex PCR assay to demonstrate its accuracy. Using the designed primers, each of the ASF, PCV2 and PPV target genes was amplified, but no other porcine virus genes were detected. The assay's limit of detection was 102 copies/µl of PCV2, PPV, or ASFV. The detection of PCV2, PPV, and ASF in postmortem tissue samples revealed that they are co-circulating in India's North-Eastern region. The percentage positivity (PP) for PCV2, PPV and ASF single infection were 7.02% (25/356), 3.93% (14/356), and 3.37% (12/356), respectively, while the PP for PCV2& PPV co-infection was 2.80% (10/356), ASF & PCV2 co infection was 1.4% (5/356) and the ASF, PPV& PCV2 co-infection was1.40% (5/356). The results also indicate that the ASF can infect pigs alongside PCV and PPV.

Development of gold Immunochromatographic assay strip based on specific polyclonal antibodies against capsid protein for rapid detection of porcine circovirus 2 in Zhejiang province, China.

BACKGROUND: The existing detection methods for porcine circovirus type 2 (PCV2) specific antibodies in serum cannot determine the infection status, thus it is necessary to establish a method for detecting PCV2 antigen. The capsid protein (CAP) of PCV2, as a major structural protein that plays a significant role in viral replication and in inducing host's immune response, is an ideal target antigen to monitor PCV2 infection. Therefore, a gold immunochromatographic assay (GICA) for rapid detection of PCV2 antigen based on the polyclonal antibodies (PAbs) against PCV2-CAP will be developed. RESULTS: The truncated CAP protein (dCAP) was used to immunize rabbits to generate anti-serum. After preliminary purification by caprylic acid/ammonium sulfate precipitation (CAAS), specific PAbs were purified by affinity chromatography column coupled with dCAP and its titer was about two-fold higher than preliminary purified PAbs. Colloidal gold-PAbs conjugate was synthesized under the optimum conditions. The specific anti-dCAP PAbs and goat anti-rabbit antibody (GAR) were then sprayed onto nitrocellulose (NC) membrane as a test line (TL) and a control line (CL), respectively. The visual limit detection (vLOD) of the GICA strips was 5 ng/mL. Specificity assay indicated that the GICA strips had specifically detected PCV2 and was not reactive for porcine epidemic diarrhea virus (PEDV), pseudorabies virus (PRV), porcine reproductive and respiratory syndrome virus (PRRSV) or classic swine fever virus (CSFV). A total of 36 porcine serum samples were detected by this GICA and commercial enzyme-linked immunosorbent assay (ELISA) Kit, 9 positive samples were found by the developed strip with the rate of 25.0% comparing with 11 positive samples detected by the commercially ELISA Kit which positive rate was 30.5%, and the receiver operating characteristic (ROC) curve revealed that the relative sensitivity and specificity of this GICA strip were 72.7 and 96.0%, respectively, with an area of 87.2%. CONCLUSIONS: This study established an efficient detection method with high sensitivity and specificity for the clinical diagnosis of PCV2 antigen, that will facilitate a rapid and convenient way to evaluate the infection status of vaccinated pigs.

Combined Antibody Tagged HRP Gold Nanoparticle Probe for Effective PCV2 Screening in Pig Farms.

Introduction: Porcine circovirus type 2 (PCV2) causes immune repression and intercurrent infections in pigs, resulting in a huge economic loss to the pig breeding industry. Additionally, the spread of PCV2 in pig farms can pollute the living environment of the residents in the farm's vicinity, which increases the rate of infections. Therefore, rapid and sensitive detection methods are needed for disease prevention and timely environmental cleaning. Methods: This research describes a highly sensitive sandwich enzyme-linked immunosorbent assay (ELISA) that utilizes gold nanoparticles (AuNPs) in a functional, specific antibody labeled probe for the detection of PCV2. Due to their high specific surface area and histocompatibility, AuNPs were used as carriers of HRP labeled anti-PCV2 antibodies to amplify the detection signal. Results: Compared to conventional sandwich ELISA procedures, this method resulted in higher sensitivity (51-fold) and a shorter assay time with a limit of detection of 195 TCID50/mL. The cross-reactivity assay demonstrated that this assay was PCV2 specific. Conclusion: The amplified Ab (HRP) labeled AuNPs probe provides a sensitive analytical approach for the determination of the traces of the PCV2 antigen in early diagnosis.

Development and Primary Application of an Indirect ELISA Based on Rep Protein to Analyze Antibodies against Porcine Cocirvirus-like Virus (PCLV).

Porcine circovirus-like virus (PCLV) is a member of circovirus that contains a single-strand DNA genome, which may be one of the pathogens that causes diarrheal symptoms in pigs. The Rep protein encoded by the genome of PCLV may be responsible for viral genome replication. The development of serological detection methods for PCLV is of great necessity for clinical diagnosis, as well as epidemiological investigations. Therefore, this study attempted to build an indirect enzyme-linked immunosorbent assay (ELISA) to examine antibodies against PCLV based on the His-tagged recombinant Rep protein. Full-length PCLV Rep protein was induced and expressed in E. coli and was purified as an antigen to establish an ELISA detection kit. The purified Rep protein was used to inject into mice to produce specific antibodies. There was no cross-reaction of Rep-based ELISA with antisera against other porcine viruses. The intra-assay and inter-assay coefficient variations (CVs) were 0.644-8.211% and 0.859-7.246%, respectively, indicating good repeatability. The non-cross-reaction with TGEV, PRRSV and PCV2 testing showed high sensitivity and high specificity for this ELISA assay. A total of 1593 serum samples collected from different pig farms in Jiangxi Province were tested for anti-PCLV Rep antibodies, and 284 (17.83%) of the 1593 samples were Rep antibody positive. Altogether, the indirect ELISA detection tool developed in this study could be applied to examine serum of PCLV antibodies with good repeatability, high sensitivity and high specificity. In addition, field sample detection results suggested that the PCLV antibody has a low prevalence in pig populations in Jiangxi Province of China.

Expression and immunogenicity analysis of the capsid proteins of porcine circovirus types 2 to 4.

Porcine circovirus (PCV) comprises four types, PCV1, PCV2, PCV3, and PCV4, which belong to the Circovirus genus of the family Circoviridae. PCV1 is nonpathogenic, whereas PCV2, PCV3, and PCV4 can infect pigs and cause disease. However, due to a lack of experimental evidence, whether vaccines based on PCV capsid (Cap) can induce cross-reactivity against PCVs remains controversial. In this study, recombinant truncated capsids (rCaps) of PCV2, PCV3, and PCV4 were highly and efficiently expressed and purified, followed by the development and evaluation of antibodies against PCVs. The results showed that monovalent and trivalent antigens based on the recombinant Caps had adequate immunogenicity to stimulate specific antibodies against the corresponding protein and virus. Furthermore, antisera prepared from the recombinant Caps also cross-reacted with different PCVs. Therefore, recombinant proteins can be used as candidate antigens to develop vaccines and ELISA diagnostic kits. In addition, the antibodies prepared in this study are promising candidates for the simultaneous prevention and treatment of PCVs in the clinic.

Development and applications of a TaqMan based quantitative real-time PCR for the rapid detection of Pigeon circovirus (PiCV).

TaqMan probe based quantitative polymerase reaction (TaqMan qPCR) is a robust and reliable technique for detecting and quantifying target DNA copies. Quantitative molecular diagnosis of genetically diverse single stranded DNA (ssDNA) virus such as Pigeon circovirus (PiCV) can be challenging owing to difficulties in primer binding or low abundance of template DNA copies in clinical specimens. Several methods have been described for the detection of PiCV, being qPCR the most simple and reliable. As far as is known, two qPCR systems described until now are based on SYBR green. This study reports development and validation of a highly sensitive TaqMan qPCR targeted to Rep for the detection of highly diverse PiCV in pigeon samples with excellent reproducibility, specificity, and sensitivity. The limit of detection was determined as low as 2 (two) plasmid copies. Estimations of 100 % specificity and 100 % sensitivity were obtained based on the qPCR results with panel of 60 samples (known PiCV positive, n = 30; known PiCV negative, n = 20; samples positive to Beak and feather disease virus (BFDV), n = 5 and samples positive to canine circovirus, n = 5). Co-efficient of variation (CV) for Ct values ranged between 0.27 % and 0.78 % in the same assay and 1.84-2.87 % in different assays.

Frequency of porcine circovirus 3 detection and histologic lesions in clinical samples from swine in the United States.

Porcine circovirus 3 (PCV3) is widespread in pigs worldwide. Diverse clinical signs and lesions have been associated with PCV3, but the role of PCV3 as a cause of disease in swine remains unclear. We investigated the association of PCV3 with clinical signs and histologic lesions in 730 diagnostic swine cases between February 2016 and January 2018. The cases contained 2,177 samples submitted from 474 sites located in 21 states in the United States. PCR assay results were positive for PCV3 for 577 of 2,177 (27%) samples, 255 of 730 (35%) cases, 181 of 474 (38%) sites, and 17 of 21 (81%) states. We detected PCV3 in 19 of 28 specimen types and in pigs of all ages and clinical presentations, including healthy pigs, with the highest detection rate in adult pigs. PCV3 detection was not associated with respiratory, gastrointestinal, or CNS signs, weight loss, or sudden death. Of 58 types of histologic lesions evaluated, PCV3 detection was associated with myocarditis, cardiac vasculitis, and interstitial pneumonia in growing pigs. A high PCV3 detection rate was observed in aborted fetuses.

Rapid screening of monoclonal antibodies against porcine circovirus type 2 using colloidal gold-based paper test.

A proof of concept for using paper test as a suitable method in the production of monoclonal antibodies (MAbs) is reported. The paper test which detects antibodies against porcine circovirus type 2 (PCV2) using colloidal gold-labelled capsid protein as the antigen probe was applied exclusively in the screening of anti-PCV2 MAbs. It allowed the detection of 118 single cell clones within 30 min using naked eyes. MAbs with specific binding to authentic epitopes on the virus were selected using a blocking strategy in which the antibody was pre-incubated with PCV2 viral sample before applying to the test paper. Five hybridomas secreting MAbs against the capsid protein were obtained, with only three of them capable of binding to PCV2. The results were validated and confirmed using enzyme-linked immunosorbent assay and immunofluorescence assay. The paper test is simple, rapid, and independent on professional technicians and proves to be an excellent approach for the screening of MAbs against specific targets.

Five years of porcine circovirus 3: What have we learned about the clinical disease, immune pathogenesis, and diagnosis.

Porcine circovirus type 3 (PCV3) is a non-enveloped, circular, single-stranded DNA virus in the family Circoviridae. This member of the genus Circovirus was initially described as affecting swine in 2016, and new research has provided further insight into its structural characteristics, disease presentations, pathogenesis, and immune response following infection. Therefore, this review aims to summarize advances in PCV3-related research about genomic characteristics epidemiology, pathogenesis, immune response, and the development of diagnostics. PCV3 has been detected globally and retrospectively in pigs of all ages and is associated with a range of clinical presentations, including multisystemic inflammatory syndrome, reproductive failure, porcine dermatitis and nephropathy syndrome, and subclinical infection. Experimental studies have successfully reproduced multisystemic inflammation but have not detected clinical disease. These findings, coupled with a large number of reports of coinfections coinciding with PCV3, may suggest that PCV3 infection alone may not be sufficient to cause evidenceable clinical disease. The pathogenesis of PCV3 has not been fully elucidated yet, and while receptors that facilitate cell-viral entry have not been identified, replication has been confirmed in a wide range of cell types, including trophoblasts, myocardiocytes, skin adipocytes, and neurons. PCV3 seems to evade the host immune response as evidenced by persistent viremia 42 days post-infection in experimental and longitudinal field studies despite a strong humoral response. Minimal differences in host cytokine profiles and peripheral cell-mediated responses have been observed, but certainly many questions still surround the mechanisms by which PCV3 evades the immune response. The epidemiology of PCV3 remains unclear, and the exact routes of transmission have not been described; but, PCV3 can be shed in oral fluids, nasal secretions, feces, colostrum, and semen, demonstrating the importance of lateral and vertical transmission. The detection of PCV3 in numerous domesticated and wild animal species, including cattle, dogs, mice, wild boar, chamois, roe deer, ticks, and mosquitoes, suggests the potential for multiple reservoirs and cross-species transmission. Current advances in PCV3 diagnostic tests have the ability to differentiate PCV3 from other PCVs and corroborate its presence within lesions. Given that the economic impact associated with PCV3 infection has not been assessed and the virus has the potential to emerge as a high-prevalence pathogen in the coming years, future research should focus on filling the knowledge gaps identified in this review.

Multiplex polymerase chain reaction for the detection and differentiation of 4 porcine circovirus 2 genotypes (PCV-2a, -2b, -2d, and -2e) in clinical samples.

The objective of this study was to develop multiplex polymerase chain reaction (PCR) for the simultaneous detection of porcine circovirus 2 (PCV-2) and differentiation among 4 PCV-2 genotypes (2a, 2b, 2d, and 2e) in collected clinical lymph node samples. The multiplex PCR detected each of 4 PCV-2 genotypes (2a, 2b, 2d, and 2e) to a dilution of 2 × 101 copies/µL. PCV-2a, PCV-2b, PCV-2d, and PCV-2e were propagated in tissues prior to DNA extraction for use in multiplex PCR for the simultaneous detection and differentiation of 4 PCV-2 genotypes. The designed multiplex PCR effectively detected and differentiated various combinations of multiple infection, such as PCV-2a+2b, PCV-2a+2d, PCV-2b+2d, PCV-2a+2e, and PCV-2a+2b+2d, in clinical lymph node samples. The results of this study demonstrated that multiplex PCR testing of clinical samples developed herein was able to simultaneously detect and differentiate among the 4 PCV-2 genotypes (PCV-2a, 2b, 2d, and 2e).

L'objectif de cette étude était de développer une réaction en chaîne par la polymérase multiplex (PCR) pour la détection simultanée du circovirus porcin 2 (PCV-2) et la différenciation entre quatre génotypes PCV-2 (2a, 2b, 2d et 2e) dans des échantillons cliniques de noeuds lymphatique. La PCR multiplex a détecté chacun des quatre génotypes PCV-2 (2a, 2b, 2d et 2e) à une dilution de 2 × 101 copies/µL. Le PCV-2a, le PCV-2b, le PCV-2d et le PCV-2e ont été propagés dans les tissus avant l'extraction de l'ADN pour être utilisés dans la PCR multiplex pour la détection et la différenciation simultanées de quatre génotypes du PCV-2. La PCR multiplex conçue a efficacement détecté et différencié diverses combinaisons d'infections multiples, telles que PCV-2a+2b, PCV-2a+2d, PCV-2b+2d, PCV-2a+2e et PCV-2a+2b+2d, dans les échantillons cliniques de ganglions lymphatiques. Les résultats de cette étude ont démontré que le test PCR multiplex des échantillons cliniques développés ici était capable de détecter et de différencier simultanément les quatre génotypes PCV-2 (PCV-2a, 2b, 2d et 2e).(Traduit par Docteur Serge Messier).