Detection Of Porcine Circovirus Type 2 Research Articles

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 anddeveloped a multiplex crystal digital PCR (cdPCR) method after optimizing the primerconcentration, 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.

Development of a recombinase-aided amplification method combined with lateral flow dipstick assay to detect Porcine circovirus type 2.

Porcine circovirus type 2 (PCV2) is a pathogenic virus that suppresses the immune system of pigs, impacting their health and causing economic losses. Rapid diagnostic tools for early detection of PCV2 are critical to disease prevention and control. Several molecular techniques have been established for detecting PCV2 but costly equipment and time-consuming methods are unsuitable for field inspection. In this study, we developed a recombinase-aided amplification combined with lateral flow dipstick (RAA-LFD) assay to compare with polymerase chain reaction (PCR) and quantitative PCR (qPCR) in detecting PCV2 in suspected field samples. To amplify RAA products, 15 primer pairs were designed from the conserved region of the open reading frame (ORF) 1 gene based on multiple alignments of eight PCV2 genotypes. The most efficient primer pair and conditions for the RAA-LFD assay were tested and selected. Limit of detection, repeatability, and reproducibility were determined using the constructed plasmid. DNA was extracted from positive samples for specificity testing as well as from 100 field samples to compare the detection of the RAA-LFD assay with PCR and qPCR. The F1/R1 primer pair was chosen and labeled with fluorescein isothiocyanate at the 5' end of the forward primer and with biotin at the 5' end of the reverse primer. The limit of detection of the RAA-LFD assay was 10 copies/μL at 38°C for 30 min. The RAA-LFD assay was repeatable and reproducible, with no cross-reaction with PCV3, Actinobacillus pleuropneumoniae, Porcine epidemic diarrhea virus, Classical swine fever virus, Porcine reproductive and respiratory syndrome virus - North America strain (PRRSV-US) and Porcine reproductive and respiratory syndrome virus - European strain (PRRSV-EU). Based on testing with 100 samples, the developed RAA showed 100% specificity and 90.56% and 85.71% sensitivity when compared to PCR and qPCR, respectively Cohen's kappa coefficients showed a good agreement with the established techniques. The RAA-LFD assay targeting the ORF1 gene was highly sensitive, specific, quick, and simple to perform in the field.

Establishment and application of multiplex real-time PCR for simultaneous detection of four viruses associated with porcine reproductive failure.

Many pathogens cause reproductive failure in sows suffering a broad spectrum of sequelae, including abortions, stillbirth, mummification, embryonic death, and infertility. Although various detection methods, such as polymerase chain reaction (PCR) and real-time PCR, have been widely used for molecular diagnosis, mainly for a single pathogen. In this study, we developed a multiplex real-time PCR method for the simultaneous detection of porcine circovirus type 2 (PCV2), porcine circovirus type 3 (PCV3), porcine parvovirus (PPV) and pseudorabies virus (PRV) associated with porcine reproductive failure. The R 2 values for the standard curve of multiplex real-time PCR of PCV2, PCV3, PPV, and PRV reached to 0.996, 0.997, 0.996, and 0.998, respectively. Importantly, the limit of detection (LoD) of PCV2, PCV3, PPV, and PRV, were 1, 10, 10, 10 copies/reaction, respectively. Meanwhile, specificity test results indicated that multiplex real-time PCR for simultaneous detection is specific for these four target pathogens and does not react with other pathogens, such as classical swine fever virus, porcine reproductive and respiratory syndrome virus, and porcine epidemic diarrhea virus. Besides, this method had good repeatability with coefficients of variation of intra- and inter-assay less than 2%. Finally, this approach was further evaluated by 315 clinical samples for its practicality in the field. The positive rates of PCV2, PCV3, PPV, and PRV were 66.67% (210/315), 8.57% (27/315), 8.89% (28/315), and 4.13% (13/315), respectively. The overall co-infection rates of two or more pathogens were 13.65% (43/315). Therefore, this multiplex real-time PCR provides an accurate and sensitive method for the identification of those four underlying DNA viruses among potential pathogenic agents, allowing it to be applied in diagnostics, surveillance, and epidemiology.

Visual and label-free ASFV and PCV2 detection by CRISPR-Cas12a combined with G-quadruplex.

African swine fever (ASF) and postweaning multisystemic wasting syndrome (PMWS) are acute infectious diseases caused by the African swine fever virus (ASFV) and porcine circovirus type 2 (PCV2). At present, there are no effective vaccines for the prevention of ASFV. PMWS, which is harmful to the domestic and even the world pig industry, is difficult to cure and has a high mortality. So, developing simple, inexpensive, and accurate analytical methods to detect and effectively diagnose ASFV and PCV2 can be conducive to avoid ASFV and PCV2 infection. CRISPR has become a potentially rapid diagnostic tool due to recent discoveries of the trans-cleavage properties of CRISPR type V effectors. Herein, we report the visual detection based on CRISPR-Cas12a (cpf1), which is more convenient than fluorescence detection. Through in vitro cleavage target DNA activation, Cas12a can trans-cleavage ssDNA G-quadruplex. TMB/H2O2 and Hemin cannot be catalyzed by cleavaged G-DNA to produce green color products. This protocol is useful for the detection of ASFV and PCV2 with high sensitivity. This method can enable the development of visual and label-free ASFV and PCV2 detection and can be carried out in the field without relying on instruments or power. This method can complete nucleic acid detection at 37 °C without using other instruments or energy. Our research has expanded the application of Cas12a and laid the foundation for the field's rapid detection of viral nucleic acid in future.

Development of Duplex LAMP Technique for Detection of Porcine Epidemic Diarrhea Virus (PEDV) and Porcine Circovirus Type 2 (PCV 2).

Porcine epidemic diarrhea virus (PEDV) and porcine circovirus type 2 (PCV2) are both important global pathogenic viruses which have a significant impact on the swine industry. In this study, a duplex loop-mediated isothermal amplification (duplex LAMP) method was developed in combination with lateral flow dipstick (LFD) for simultaneous detection of PEDV and PCV2 using specific sets of primers and probes designed based on the conserved regions of a spike gene (KF272920) and an ORF gene (EF493839), respectively. The limit of detection (LOD) values of the duplex LAMP-LFD for the detection of PEDV and PCV2 were 0.1 ng/µL and 0.246 ng/µL, respectively. The LOD of duplex LAMP-LFD was 10-times more sensitive than conventional PCR and RT-PCR-agarose gel-electrophoresis (PCR-AGE and RT-PCR-AGE). No cross-reaction to each other and to other pathogenic viruses that can infect pigs were observed according to analytical specificity tests. The duplex LAMP-LFD method for the simultaneous detection of PEDV and PCV2 co-infection could be completed within approximately 1.5 h, and only a simple heating block was required for isothermal amplification. The preliminary validation using 50 swine clinical samples with positive and negative PEDV and/or PCV2 revealed that the sensitivity, specificity, and accuracy of duplex LAMP-LFD were all 100% in comparison to conventional PCR and RT-PCR. Hence, this study suggests that duplex LAMP-LFD is a promising tool for the early detection and initial screening of PEDV and PCV2, which could be beneficial for prevention, planning, and epidemiological surveys of these diseases.

Rapid detection of porcine circovirus type 2 by a red latex microsphere immunochromatographic strip.

To establish a rapid and specific antigen detection method for porcine circovirus type 2 (PCV2), monoclonal antibodies (mAbs) were produced against the PCV2 epidemic strains and a red latex microsphere immunochromatographic strip was established. A total of eight anti-PCV2b and four anti-PCV2d mAbs were produced, and seven mAbs were confirmed to react with PCV2a, PCV2b, and PCV2d strains using an immunoperoxidase monolayer assay. The results of micro-neutralization tests showed that the mAbs 2C8, 9H4, 10G7, 7B9, and 7C7 had good neutralizing activity, whereas the neutralizing activity of the mAbs 4B3, 4C9, 6H9, and 7E2 was lower than 50%. Three mAbs, 4B3, 7C7, and 9H4, and PCV2 pAb were selected for the establishment of a red latex microsphere immunochromatographic strip, and the combination of mAb 7C7 labeled with red latex microspheres and mAb 9H4 exhibited the greatest detection ability. The immunochromatographic strip had minimum detection limits of 102.5 TCID50/0.1 ml, 100.7 TCID50/0.1 ml, and 101.5 TCID50/0.1 ml for PCV2a/CL, PCV2b/MDJ, and PCV2d/LNHC, respectively. Furthermore, no cross-reactivity was found for African swine fever virus, classical swine fever virus, porcine respiratory and reproductive syndrome virus, porcine parvovirus, porcine pseudorabies virus, porcine circovirus type 1, transmissible gastroenteritis virus, porcine epidemic diarrhea virus, porcine rotavirus, or porcine deltacoronavirus using the immunochromatographic strip. Using PCR as a reference standard, the detection sensitivity, specificity, and overall coincidence rate of the immunochromatographic strip were 81.13%, 100%, and 90.00%. Additionally, the detection ability of the immunochromatographic strip was correlated with that of virus titration. The immunochromatographic strip was used to detect 183 clinical disease samples, and the average positive detection rate was 22.95%. In summary, this method has good sensitivity and specificity and is simple, convenient, and quick to operate. It has high application value for on-site diagnosis of PCV2 and virus quantification.Key points• A red latex microsphere immunochromatographic strip for PCV2 detection was developed.• The method was not only simple to operate, but also takes less time.• The method had good sensitivity and specificity.

Development of a loop-mediated isothermal amplification (LAMP) assay for rapid visual detection of porcine circovirus type 2 (PCV2) and its application.

A cost effective, simple and rapid method is critical for detection of porcine circovirus type 2 (PCV2) infection in pigs. The present study reports the development and evaluation a loop-mediated isothermal amplification (LAMP) assay for rapid visual detection of PCV2 in pigs. The time and temperature conditions for amplification of PCV2 genes were optimized to be 30 min at 67 °C. The developed assay was 10 fold more sensitive than conventional PCR with analytical sensitivity of 5 pg and 50 pg, respectively. The developed LAMP assay had a sensitivity of 100%, specificity of 85.45% and overall accuracy of 89.70%. This is perhaps the most rapid of all LAMP reports for PCV2 detection available globally. The assay did not cross-react with porcine parvovirus or classical swine fever virus. DNA sequencing was done to ensure accuracy of LAMP assay results. The assay was assembled into a kit of 20 reactions and validated in different laboratories in India. The developed LAMP assay was proved to be a specific, sensitive and rapid method for visual detection of PCV2 which does not require costly equipments.

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

IntroductionPorcine 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.MethodsThis 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.ResultsCompared 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.ConclusionThe amplified Ab (HRP) labeled AuNPs probe provides a sensitive analytical approach for the determination of the traces of the PCV2 antigen in early diagnosis.

Prevalence of porcine parvovirus 1 through 7 (PPV1-PPV7) and co-factor association with PCV2 and PRRSV in Korea

BackgroundClassical porcine parvovirus (PPV1) and novel porcine parvoviruses designated porcine parvovirus 2 through 7 (PPV2-PPV7) are widespread in pig populations. The objective of this study was to investigate the prevalence rates of PPV1-PPV7 in Korea by detecting PPVs in serum, lung and fecal samples and to elucidate the association of PPVs with porcine circovirus type 2 (PCV2) and porcine reproductive and respiratory virus (PRRSV), major pathogens involved in porcine respiratory disease complex (PRDC). A total of 286 serum, 481 lung, and 281 fecal samples collected from 2018 to 2020 were analyzed.ResultsThe results showed that PPVs are widespread in Korea; the highest detection rates were found in lung samples and ranged from 7.9% (PPV1) to 32.6% (PPV2). Regarding age groups, fattening pigs had the highest detection rates of PPVs, ranging from 6.4% (PPV1) to 36.5% (PPV6); this finding suggests the chronic nature of PPV infections and the continual circulation of these viruses. When compared with PCV2- and PRRSV-negative lung samples, PCV2-positive samples with or without PRRSV positivity had significantly higher detection levels of PPV1 and PPV6. In contrast, the prevalence of PPV2 and PPV7 was significantly higher in PRRSV-infected lung samples regardless of PCV2 detection. PPV5 was detected significantly more frequently in samples with both PCV2 and PRRSV positivity.ConclusionsThis study could offer a better understanding of the role of PPVs in PCV2 and/or PRRSV infection though further studies are needed to experimentally assess the impact of PPVs in coinfections.

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.

Selection and Characterization of a Single-Chain Variable Fragment against Porcine Circovirus Type 2 Capsid and Impedimetric Immunosensor Development.

Porcine circovirus type 2 (PCV2) is the primary causative agent of porcine circovirus-associated disease (PCVAD) that causes huge global economic losses for the swine industry. Effective strategies or rapid detection of PCV2 in pig are essential to control PCVAD. Here, single-chain variable fragments (scFvs) were selected and characterized against the PCV2 capsid using phage display technology. Phage scFv clones were selected from the human scFv phagemid library (Tomlinson I + J) for direct panning against the PCV2 capsid. Eighty-four monoclonal phage scFvs were individually tested for binding to the PCV2 capsid by ELISA. Eight scFv clones showed significant binding to the PCV2 capsid and only three clones (clone nos. 13, 37, and 81) contained both VHCDRs and VLCDRs in the sequence. Clone scFv no. 81 had the highest reactivity to the PCV2 capsid and was constructed in the pET22b (+) expression vector. The recombinant was transformed to Escherichia coli BL21(DE3) for expression and purification. The scFv showed appropriate affinity to the PCV2 capsid by western blot analysis. Kinetics of scFv and the PCV2 capsid were determined using surface plasmon resonance and showed binding affinity in the nanomolar range (KD = 57.2 nM). Our scFv was first applied in the development of an impedimetric immunosensor for PCV2 capsid detection, and results showed that impedance increased with increasing PCV2 capsid expression with limit of detection = 114 nM. Findings demonstrated that our scFv has potential for use as a receptor for biosensor devices.

Development of Recombinase Aided Amplification Combined With Disposable Nucleic Acid Test Strip for Rapid Detection of Porcine Circovirus Type 2.

Porcine circovirus type 2 (PCV2) is the dominant causative agent of PCV2 systemic disease (PCV2-SD) in pigs. It can also associate with other diseases such as respiratory and enteric diseases, reproductive failure, porcine dermatitis and nephropathy syndrome in pigs. Currently, PCV2 infection is a considerable threat in the swine industry. Therefore, it is of great significance to prevent, control, and accurately detect PCV2 in pig farms. Recombinase aided amplification (RAA) technology is an isothermal nucleic acid amplification technology that could rapidly amplify the target gene fragment at a constant temperature. The amplification products labeled with specific molecules could be visually detected using the test strip with the corresponding antibody. In the present study, the RAA technology combined with a nucleic acid test strip (RAA-strip) was established for simple and specific detection of PCV2. Primers and probes targeting the PCV2 ORF2 gene were designed according to the RAA technology principles. The PCV2 RAA-strip established in this study could detect as low as 103 copies/μL of recombinant plasmids containing the PCV2 ORF2 gene fragment. The lowest detection limit about viral DNA and virus titers was 6.7 × 10−6 ng/μL and 10 TCID50/mL, respectively. Furthermore, no cross-reaction with other porcine viruses occurred at 37°C and within 15 min. We used 42 clinical samples to assess the performance of our established method. The positive rate of clinical samples detected by PCV2 RAA-strip was 50.00%. This was similar to that detected by PCV2 PCR (45.24%). In conclusion, due to the advantages of strong specificity, high sensitivity, excellent reproducibility, and simple operation method, our PCV2 RAA-strip is suitable for the rapid clinical detection of PCV2 on-site.

Applicability of Raman spectroscopy on porcine parvovirus and porcine circovirus type 2 detection

Porcine parvovirus (PPV) is one of the major infectious causes of reproductive failure of swine. This disease is characterized by embryonic and fetal infection and death, responsible for important economic losses. PPV is also implicated as a trigger in the development of post-weaning multisystemic wasting syndrome (PMWS) caused by Porcine circovirus type 2 (PCV2). Their detection is PCR-based, which is quite sensitive and specific, but laborious, costly and time-demanding. Therefore, this study aimed to assess Raman spectroscopy (RS) as a diagnostic tool for PPV and PCV2 due to its label-free properties and unique ability to search and identify molecular fingerprints. Briefly, swine testis (ST) cells were inoculated with PPV or PCV2 and in vitro cultured (37 °C, 5% CO2) for four days. Fixed cells were then submitted to RS investigation using a 633 nm laser. A total of 225 spectra centered at 1300 cm−1 was obtained for each sample (5 spectra/cell; 15 cells/replicate; 3 replicates) of PPV-, PCV2-infected and uninfected (control) ST cells. Clear statistical discrimination between samples from both virus-infected cells was achieved with a Principal Component – Linear Discriminant Analysis (PCA-LDA) model, reaching sensitivity rates from 95.55% to 97.77%, respectively to PCV2- and PPV-infected cells. These results were then submitted to a Leave-One-Out (LOO) validation algorithm resulting in 99.97% of accuracy. Extensive band assignment was analyzed and compiled for better understanding of PPV and PCV2 virus-cell interaction, demonstrating that specific protein, lipids and DNA/RNA bands are the most important assignments related to discrimination of virus-infected from uninfected cells. In conclusion, these results represent promising bases for RS application on PCV2 and PPV detection for future diagnostic applications.

Comparison of Multiplex Real-Time PCR and PCR-Reverse Blot Hybridization Assays for the Direct and Rapid Detection of Porcine Circovirus Type 2 Genotypes

Porcine circovirus type 2 (PCV2), the causative agent of porcine circovirus-associated diseases (PCVAD), poses a serious economic threat for the swine industry. Currently, PCV2 is classified into five major genotypes: PCV2a, PCV2b, PCV2c, PCV2d, and PCV2e. The aim of this study is to evaluate the performance of two commercially available methods, multiplex real-time PCR assay and PCR-reverse blot hybridization assay (REBA), for the rapid detection of PCV2 and direct identification of PCV2 genotypes from clinical samples as well as to compare the results with that of sequence analysis. Molecular diagnostic methods were used to evaluate a total of 180 samples, including tissues and blood samples from pigs that were suspected of PCVAD infection. The results of this study showed that the detection rate for positive PCV2 was 48.3% (n = 87) in both multiplex real-time PCR and PCR-REBA methods. Using sequence analysis, which is the gold standard, and multiplex real time PCR assay, the sensitivity, specificity, positive predictive value, and negative predictive value of PCV2 genotyping were found to be 97.1% (n = 67, 95% CI 0.894–0.998, p < 0.001), 100% (n = 93, 95% CI 0.966–1.000, p < 0.001), 100% (95% CI 0.953–1.000, p < 0.001), 97.9% (95% CI 0.921–0.998, p < 0.001), respectively. The results of PCR-REBA were found to be consistent with those of sequence analysis for all the samples and showed good agreement (κ = 1). The most prevalent genotypes detected in this study were PCV2d (n = 53, 60.9%), followed by PCV2a (n = 17, 19.5%), PCV2b (n = 14, 16.1%), and PCV2a/b co-infection (n = 3, 3.5%). Both the methods required ~3 h for completion. Therefore, we conclude that two molecular methods are rapid and reliable for the characterization of the causative pathogen with PCV2 genotypes.

Do porcine parvoviruses 1 through 7 (PPV1-PPV7) have an impact on porcine circovirus type 2 (PCV2) viremia in pigs?

Infections with porcine parvoviruses 1 through 7 (PPV1-PPV7) and porcine circovirus type 2 (PCV2) are widespread in pig population. PCV2 is involved in a number of disease syndromes collectively called PCV2‐associated diseases (PCVD). It is well elucidated, that PPV1 may act as a triggering factor of PCVD through supporting PCV2 replication. Less is known about the PPV2-PPV7 impact on PCV2 viremia, but several authors suggested an association between these viruses. In order to provide a better understanding of PCV2 and PPVs co-infections, 519 serum samples from eight Polish swine farms were tested by real-time PCR to assess the possible impact of PPV1-PPV7 on PCV2 viremia. Among all 519 serum samples, 30.6 % were positive for PCV2 and PPVs detection rates ranged from 2.9 % (PPV1) to 26.6 % (PPV2). Within 159 serum samples categorized as PCV2-positive, the prevalence rates of PPVs ranged from 7.5 % (PPV1) to 37.1 % (PPV6). The level of PCV2 viremia was significantly higher only in serum samples positive for PPV1 and PPV7 compared to samples negative for these PPVs. Moreover, the correlation between Ct values for PPV7 and PCV2 was observed. Thus, our results suggested that apart from PPV1, also PPV7 stimulate the replication of PCV2.

Real-Time PCR Detection Patterns of Porcine Circovirus Type 2 (PCV2) in Polish Farms with Different Statuses of Vaccination against PCV2.

Porcine circovirus type 2 (PCV2) is a globally spread pathogen controlled with generally highly efficacious vaccination protocols. In order to compare PCV2 detection profiles in farms with different vaccination statuses, serum (359) and fecal pools (351) and oral fluids (209) from four farms that do not vaccinate against PCV2 (NON-VAC) and from 22 farms that do vaccinate (VAC) were tested with quantitative real-time PCR. Additionally, nucleotide sequences of ORF2 of the virus were obtained from selected samples. Three genotypes, PCV2a, PCV2b, and PCV2d, were detected. Significant differences (p < 0.05) in PCV2 prevalence and quantities between the VAC and NON-VAC farms were evident. In five VAC farms, no viremia or shedding in feces was detected. On the other hand, in four VAC farms, the results were very similar to those from NON-VAC farms. No significant difference in PCV2 prevalence in oral fluids was observed between VAC and NON-VAC farms. An examination of viremia can be recommended for the detection of vaccination efficacy issues. The median of the PCV2 viral loads >6.0 log10 copies/mL in pooled sera from the vaccinated population should be considered a very strong indication that the vaccination protocol needs revision.

Highly Sensitive Detection of PCV2 Based on Tyramide Signals and GNPL Amplification.

The frequent emergence of secondary infection and immunosuppression after porcine circovirus type 2 (PCV2) infection highlights the need to develop sensitive detection methods. A dual-signal amplification enzyme-linked immunosorbent assay (ELISA) based on a microplate coated with gold nanoparticle layers (GNPL) and tyramide signal amplification (TSA) was established. Results confirmed that the microplates coated with GNPL have a strong binding ability to the antibody without affecting the biological activity of the antibody. The microplates coated with GNPL have strong binding ability to the antibody, and the amplification of the tyramide signal is combined to further improve the sensitivity of PCV2. The PCV2 antibody does not crossreact with other viruses, demonstrating that the method has good specificity. A dual-signal amplification strategy is developed using microplates modified with GNPL and TSA to sensitively detect PCV2.

Porcine circovirus type II screening in feral swine population in Ukraine

Porcine circovirus type 2 (PCV2) is an emergent single-stranded DNA virus found all over the world in domestic pigs and wild boars that causes infectious disease with a great impact on swine productivity. PCV2 has 1.7 kb genome that includes two main genes, which encode replication-related protein (rep) and the major structural capsid (cap) protein. Both of these genes can be used as target sequences for the primer design for the detection of PCV2 as well as for sequencing of designated regions. We carried out a screening due to the PCV2 circulating among the wild boar population in 10 regions of Ukraine. PCR screening was performed using primer pairs designed on the target sequences of the replicative and capsid genes. According to the results of the research, the presence of genetic material of PCV2 was found in 31.8% of the tested samples. The developed set of primers may be suitable for diagnostics, as well as for the development of specific sites for the purpose of sequencing of PCV2 cap-gene due to the obtained DNA samples during epizootic screening

Development and validation of a real-time PCR for the detection and quantification of porcine circovirus type 2.

Porcine circovirus type 2 (PCV2) is a spherical and non-enveloped virus belonging to the genus Circovirus of the Circoviridae family with a single stranded circular DNA genome. This virus, already detected worldwide, has been associated to several diseases and was implicated as the etiological agent of a disease named postweaning multisystemic wasting syndrome. Several methods have been described for the detection of PCV2, being real-time PCR the most simple and reliable. As far as we know, all the real-time PCR systems described until now are based on ORF2 gene, that exhibit the highest variability. This paper reports the development and validation of a real-time PCR targeted to ORF1 and based on a TaqMan probe for the detection of porcine circovirus type 2 DNA in swine samples. Due to the lack of PCV1 samples, the ability of the test to discriminate between PCV1 and PCV2 positive samples was evaluated in silico. Estimations of 100% specificity and 100% sensitivity were obtained based on the qPCR results with panel of 81 swine samples (known PCV2-positive (n = 50); known PCV2-negative (n = 17); samples positive to other common swine viral pathogens (n = 13) and one sample from a BFDV-positive parrot (n = 1)). Intra- and inter-assay coefficients of variation obtained with three positive samples of different viral charges in five replicates or in five independent assays were below the acceptance threshold. The limit of detection determined with a recombinant plasmid containing the amplicon, led to conclude that this assay can detect at least three plasmid copies.

Genetic characterization of porcine circovirus type 2 in captive wild boars in southern Brazil.

Porcine circovirus type 2 (PCV2) has been identified in pig population in Brazil since 2000, but scarce studies involving wild boars with PCV2 infection are reported in the country. This study aimed to perform the genetic characterization of PCV2 detected in clinically healthy captive wild boars from farms located in Southern Brazil. Bronchial and mesenteric lymph nodes from 129 clinically healthy captive wild boars were tested by nested PCR for PCV2 detection. Six out of 38 positive samples (29.5%) were submitted to a quantitative real time PCR (qPCR) and genetic sequencing. Viral load up to 1.19×109 viral DNA copies/uL was detected in lymph nodes samples by qPCR. According to the ORF2 gene sequence analysis, all PCV2 samples were classified into PCV2b genotype. Comparisons based on a 702nt region of the ORF2 of all six isolates revealed a high degree of similarity between these isolates. The ORF2 sequences characterized here share 97.1-100% of nucleotide identity and 95.7-100% of amino acid identity with other PCV2b isolated in Brazil from wild boars and feral pigs. This study reports the first detection and genetic characterization of PCV2b in captive wild boars in Brazil and provides important information on PCV2 infection in this domesticated species.