PCV2 Infection Research Articles

Emergence of novel Porcine circovirus 2 genotypes in Southern India.

A total of 200 samples from Porcine circovirus 2 suspected (n = 112) and healthy (n = 88) swine populations collected from different districts of Tamil Nadu, south India were used in this study. The samples comprising of serum (n = 124), swabs from natural orifices (n = 52), and postmortem tissues (n = 24). All the samples were processed and subjected to the screening and detection of the PCV2 genome by a specific PCR assay. PCV2 genomes from positive samples were further subjected to genotyping with specifically designed primers for the full-length amplification of the ORF2 gene which codes for capsid protein (Cp) and serves as an epidemiological marker. Randomly, 13 amplified ORF2 genes were sequenced and the aligned sequences were subjected to signature motif analysis and phylogeny in MEGA X. The molecular prevalence of PCV2 infection in Tamil Nadu is 10.5% (n = 21). Signature motif and phylogenetic studies of 13 samples revealed 38.5% (n = 5) presence of each PCV2b intermediate 1(IM1) and PCV2b genotypes, followed by 15.4% (n = 2) PCV2d-2 and 7.7% (n = 1) PCV2d genotypes. The PCV2b-IM1 genotype has a 99.43% sequence homology with Vietnam isolate (JX506730). PCV2b genotypes showed 99.72% sequence identity with Chinese isolate (KX068219). PCV2d-2 genotypes reported in this study have 100% sequence identity with Taiwan isolate (MF169721). PCV2d genotype showed 97.87% sequence identity with Thailand isolate (MF314293). Amino acid analysis of all the 13 full-length ORF2 gene sequences revealed specific mutations in the immune reactive domains of A, B, C, and D. Capsid protein of three PCV2b and five PCV2b IM1 isolates had extra amino acid residue lysine (K) at 234 position of ORF2 similar to PCV2d. For the first time in South India, PCV2b IM1 and PCV2d-2 genotypes are reported. This study evidences the genetic shifts of PCV2 isolates in India and it is analogous to that of global genotypic shift.

Retrospective surveillance of porcine circovirus 4 in pigs in Inner Mongolia, China, from 2016 to 2018.

A novel circovirus designated "porcine circovirus type 4" (PCV4) was recently reported in pigs with severe clinical disease in Hunan Province, China. Relatively little is known about the molecular epidemiology of this recently discovered virus. In order to assess the prevalence of PCV4 infection in pigs and to analyze its genomic characteristics, 1683 clinical samples were collected in Inner Mongolia, China, from 2016 to 2018. The overall infection rate of PCV4 was 1.6% (27/1683) at the sample level and 21.6% (11/51) at the farm level, with rates ranging from 3.2% (1/31) to 20.0% (6/30) on different PCV4-positive pig farms. In addition, the PCV4 infection rates at both the sample and farm level increased from 2016 to 2018. This also showed that PCV4 was present in pigs in 2016 in China and therefore did not arrive later than this date. Additionally, our findings showed that PCV4 infections had no association with PCV2 or PCV3 infections. We sequenced the complete genomes of three PCV4 strains and found that the PCV4 strains had a high degree of genetic stability but shared less than 80% sequence identity with other circoviruses. We identified six amino acid mutations in the Rep protein and seven in the Cap protein. Phylogenetic analysis based on Cap and Rep sequences confirmed that the PCV4 strains grouped in an independent branch. Our findings provide important information about the prevalence and genetic characteristics of PCV4 strains.

A 14 bp indel polymorphism in the promoter region is associated with different responses to porcine circovirus type 2 infection by regulating MRC1 transcription

Mannose receptor, C type 1 (MRC1) is a key factor in regulating the body's immune response to resist pathogen invasions. In this study, mRNA expressions of MRC1 gene in nine porcine organs/tissues were compared between Laiwu (LW) and Yorkshire × Landrace crossbred (YL) pigs prior to and post PCV2 infection. We found that, for pigs uninfected with PCV2, MRC1 mRNA expressions in the lung, spleen, large intestine, small intestine and mesenteric lymph node tissues of LW were significantly higher than those of YL pigs (P < 0.05). After PCV2 infection, MRC1 mRNA levels in the liver, kidney and mesenteric lymph node were significantly increased in LW pigs (P < 0.05); while, significantly decreased in the heart and lung tissues of YL pigs (P < 0.05). The transcriptional activity of porcine MRC1 promoter was further analyzed to investigate the molecular mechanism underlying these expressional differences in response to PCV2 infection. Luciferase assay indicated that a 14 bp indel polymorphism “GTTTTTTTTTTTTT” at the site −864 of MRC1 promoter contributed to the transcriptional activity. The frequency of 14 bp insertion in LW and Dapulian pigs, generally resistant to PCV2 infection, was higher than that in Duroc, Landrace and Yorkshire pigs, which were sensitive to PCV2 infection. The promoter with 14 bp insertion displayed higher MRC1 transcription level both prior to and post PCV2 infection compared with that carrying no insertion in PK15 cells (P < 0.01). The results suggest that this 14 bp indel polymorphism is associated with different responses to PCV2 infection by regulating MRC1 transcription.

Porcine circovirus type 2 exploits cap to inhibit PKR activation through interaction with Hsp40

Porcine circovirus type 2 is the main pathogen of porcine circovirus disease, which has caused enormous economic losses to the pig industry worldwide. The PKR signaling pathway is important for the cellular antiviral response, but its role in the process of PCV2 infection is unknown. In this study, we first found that dsRNA was produced and that PKR was activated in PCV2 infection. However, interestingly, the activation of PKR was inhibited when the Cap protein was exogenously expressed in PAMs, and this inhibition was reversed by the expression of DNAJC7. The interaction between Cap and DNAJC7 was confirmed by laser confocal microscopy, coimmunoprecipitation and GST pull-down, and it was found that PCV2 infection or the expression of Cap protein could induce DNAJC7 to migrate to the nucleus and release P58IPK, an inhibitor of PKR activation. Downregulating the expression of DNAJC7 by a specific inhibitor or recombinant lentivirus-mediated shRNA, inhibited the replication of the PCV2 genome and the production of virions, which was consistent with the increase of DNAJC7 expression in multiple tissues of weaned piglets infected with PCV2. These data indicate that although PKR was activated by PCV2 infection, the activation was inhibited by Cap through an interaction with DNAJC7. These results help to understand the molecular mechanism of immune escape after PCV2 infection.

The combined usage of Matrine and Osthole inhibited endoplasmic reticulum apoptosis induced by PCV2

BackgroundPorcine circovirus type 2 (PCV2) is an important and common DNA virus that infect pig and can cause immunosuppression and induce apoptosis in the infected cells. To escape the host immune system, PCV2 constantly builds up complex mechanisms or mutates genes, and that is why it is difficult to eradicate complex PCV2 infection by relying on vaccines and single compound. At present, there is few literature reports on the effective prevention and treatment of PCV2 infection by a combination of two or more compounds. Previously, we have demonstrated the anti-PCV2 effect of Matrine in vitro, but its mechanism has not been further evaluated. Literatures have proven that Osthole has a variety of pharmacological activities, and we tested the ability of Osthole to inhibit PCV2 replication in cell culture. Therefore, this study explored the synergistic antiviral effect of Matrine combined with Osthole and their synergistic anti-apoptotic mechanism.ResultsOsthole alone had an anti-PCV2 effect, and then its synergistic anti-PCV2 effect of Osthole and Matrine was better than that of Matrine or Osthole alone as demonstrated by qRT-PCR, IFA and Western blotting results. The anti-apoptotic mechanism of these two compounds by inducing the PERK pathway by PCV2 was elucidated through Annexin V-FITC/PI, JC-1 and Western blotting. Matrine and Osthole combination could inhibit the expression of Cap in Cap-transfected PK-15 cells, thus inhibiting Cap-induced PERK apoptosis. Ribavirin was used as a positive control.ConclusionsThe combination of Osthole and Matrine had the synergistic effect of anti-PCV2 infection by directly inhibiting the expression of PCV2 Cap protein. The combination of these two compounds also inhibited PERK apoptosis induced by PCV2 Cap protein, possibly by regulating the level of GRP78. The results formed a base for further studies on the mechanism of anti-PCV2 in vivo using Matrine and Osthole combination and developing new anti-PCV2 compounds with Cap and GRP78 as therapeutic targets.

Construction of polycistronic baculovirus surface display vectors to express the PCV2 Cap(d41) protein and analysis of its immunogenicity in mice and swine

To increase expression levels of the PCV2 Cap(d41) protein, novel baculovirus surface display vectors with multiple expression cassettes were constructed to create recombinant baculoviruses BacSC-Cap(d41), BacDD-2Cap(d41), BacDD-3Cap(d41), and BacDD-4Cap(d41). Our results reveal that the recombinant baculovirus BacDD-4Cap(d41) was able to express the highest levels of Cap(d41) protein. Optimum conditions for expressing the PCV2 Cap(d41) protein were determined, and our results show that 107 of Sf-9 infected with the recombinant baculovirus BacDD-4Cap(d41) at an MOI of 5 for 3 days showed the highest level of protein expression. Mice immunized with the 4Cap(d41) vaccine which was prepared from the recombinant baculovirus-infected cells (107) elicited higher ELISA titers compared to the Cap (d41) vaccine. The 4Cap(d41) vaccine could elicit anti-PCV2 neutralizing antibodies and IFN-γ in mice, as confirmed by virus neutralization test and IFN-γ ELISA. Moreover, the swine lymphocyte proliferative responses indicated that the 4Cap(d41) vaccine was able to induce a clear cellular immune response. Flow cytometry analysis showed that the percentage of CD4+ T cells and CD4+/CD8+ ratio was increased significantly in SPF pigs immunized with the 4Cap(d41) vaccine. Importantly, the 4Cap(d41) vaccine induced an IFN-γ response, further confirming that its effect is through cellular immunity in SPF pigs. An in vivo challenge study revealed that the 4Cap(d41) and the commercial vaccine groups significantly reduce the viral load of vaccinated pigs as compared with the CE negative control group. Taken together, we have successfully developed a 4Cap(d41) vaccine that may be a potential subunit vaccine for preventing the disease associated with PCV2 infections.

PCV cap proteins fused with calreticulin expressed into polymers in Escherichia coli with high immunogenicity in mice

BackgroundPorcine circovirus type 2 (PCV2) is the main causative agent of porcine circovirus diseases (PCVDs) which causes huge yearly economic losses in the swine industry. Capsid protein (Cap) is the major structural protein of PCV2 that can induce a protective immune response. Therefore, developing a novel and safe subunit vaccine against PCV2 infection is needed.ResultsIn this study, the Cap gene was bound to the truncated calreticulin (CRT) (120–250 aa/120–308 aa) at the N/C terminal, and then the CRT-Cap fusion genes were expressed in Escherichia coli (E.coli). The size-exclusion chromatography and dynamic light scattering (DLS) data showed that the purified recombinant CRT-Cap fusion protein (rP5F) existed in the form of polymers. Immunization with rP5F stimulated high levels of PCV2 specific antibody and neutralization antibody in mice, which were almost identical to those induced by the commercial subunit and inactivated vaccines. The lymphocyte proliferation and cytokine secretion were also detected in rP5F immunized mice. According to the results of PCV2-challenge experiment, the virus loads significantly decreased in mice immunized with rP5F. The data obtained in the current study revealed that rP5F had the potential to be a subunit vaccine candidate against PCV2 in the future.ConclusionsWe have successfully expressed Cap-CRT fusion proteins in E.coli and optimized rP5F could form into immunogenic polymers. Mice immunized with rP5F efficiently induced humoral and part of cellular immune responses and decreased the virus content against PCV2-challenge, which suggested that rF5P could be a potential subunit vaccine candidate.

Matrine exhibits antiviral activity in a PRRSV/PCV2 co-infected mouse model

BackgroundPRRSV and PCV2 co-infection is very common in swine industry which results in huge economic losses worldwide. Although vaccination is used to prevent viral diseases, immunosuppression induced by PRRSV and PCV2 leads to vaccine failure. PurposeOur previous results have demonstrated that Matrine possess antiviral activities against PRRSV/PCV2 co-infection in vitro. This study aims to establish a PRRSV/PCV2 co-infected KM mouse model and evaluate the antiviral activities of Matrine against PRRSV/PCV2 co-infection. Study designA total of 144 KM mice were randomly divided into six groups with 24 mice in each group, named as: normal control, PRRSV/PCV2 co-infected group (PRRSV/PCV2 group), Ribavirin treatment positive control (Ribavirin control) and Matrine treatment groups (Matrine 40 mg/kg, Matrine 20 mg/kg and Matrine 10 mg/kg). MethodsExcept normal control group, all mice in other five groups were inoculated with PRRSV, followed by PCV2 at 2 h later. At 7 days post-infection (dpi), mice in the treatment groups were intraperitoneally administered with various doses of Matrine and Ribavirin, twice a day for 5 consecutive days. ResultsPRRSV N and PCV2 CAP genes were detected by PCR in multiple tissues including heart, liver, spleen, lungs, kidneys, thymus and inguinal lymph nodes. The viral load of PCV2 was the highest in liver followed by thymus and spleen. Although PRRSV were detected in most of tissues, but the replication of PRRSV was not significantly increased, as shown by qPCR analysis. Comparing with PCV2 infection alone, PRRSV infection significantly elevated PCV2 replication and exacerbated PCV2 induced interstitial pneumonia. qPCR analysis demonstrated 40 mg/kg Matrine significantly attenuated PCV2 replication in liver and alleviated virus induced interstitial pneumonia, suggesting Matrine could directly inhibit virus replication. In addition, Matrine treatment enhanced peritoneal macrophages phagocytosis at 13 and 16 dpi, and 40 mg/kg of Matrine increased the proliferation activity of lymphocytes. Body weight gain was continuously promoted by administrating Matrine at 10 mg/kg. ConclusionMatrine possessed antiviral activities via inhibiting virus replication and regulating immune functions in mice co-infected by PRRSV/PCV2. These data provide new insight into controlling PRRSV and PCV2 infection and support further research for developing Matrine as a new possible veterinary medicine.

Prevalence, pathogenesis, and evolution of porcine circovirus type 3 in China from 2016 to 2019

Porcine circovirus type 3 (PCV3) infection causes substantial economic losses in pig populations since its discovery in 2016. However, PCV3 molecular epidemiology remains need further study. In order to assess the prevalence of PCV3 infection in China, 4094 clinical samples from 271 pig farms in 10 provinces of China were evaluated by PCR. It was shown that the overall prevalence of PCV3 infection was 29.3 % (1200/4094) and 74.2 % (201/271) at sample and farm levels respectively, suggesting that PCV3 infection is prevalent in China. Furthermore, a statistical analysis showed PCV3 might exacerbate PCV2 and PRRSV infection rate and have a potential association with pig clinical disease. In addition, we sequenced the entire genome of 57 PCV3 strains; homology analysis showed that PCV3 strains have more than 96 % similarities at the nucleotide level, and PCV3 shares less than 60 % similarities with other circoviruses. By comparing the total 673 PCV3 strains from the NCBI GenBank, we found the major of amino acid mutations are located in predicted epitope regions and the mutations ratio changed during PCV3 evolution. Phylogenetic analysis revealed that all isolates belonged to PCV3a and PCV3b, and increasing PCV3a and decreasing PCV3b trends were observed during PCV3 evolution. Overall, this study provides important insights for understanding PCV3 prevalence, pathogenesis, and evolution and will guide future efforts to develop effective preventive and control measures.

Protein Interactions Network of Porcine Circovirus Type 2 Capsid With Host Proteins.

Virus-host interaction is a tug of war between pathogenesis and immunity, followed by either activating the host immune defense system to eliminate virus or manipulating host immune control mechanisms to survive and facilitate virus propagation. Comprehensive knowledge of interactions between host and viral proteins might provide hints for developing novel antiviral strategies. To gain a more detailed knowledge of the interactions with porcine circovirus type 2 capsid protein, we employed a coimmunoprecipitation combined with liquid chromatography mass spectrometry (LC-MS) approach and 222 putative PCV2 Cap-interacting host proteins were identified in the infected porcine kidney (PK-15) cells. Further, a protein-protein interactions (PPIs) network was plotted, and the PCV2 Cap-interacting host proteins were potentially involved in protein binding, DNA transcription, metabolism and innate immune response based on the gene ontology annotation and Kyoto Encyclopedia of Genes and Genomes database enrichment. Verification in vitro assay demonstrated that eight cellular proteins, namely heterogeneous nuclear ribonucleoprotein C, nucleophosmin-1, DEAD-box RNA helicase 21, importin β3, eukaryotic translation initiation factor 4A2, snail family transcriptional repressor 2, MX dynamin like GTPase 2, and intermediate chain 1 interacted with PCV2 Cap. Thus, this work effectively provides useful protein-related information to facilitate further investigation of the underlying mechanism of PCV2 infection and pathogenesis.

Chimeric VP1-PCV2bCap protein as antigen for construction of recombinant vaccine against porcine circovirus type 2

Abstract Porcine circovirus type 2 (PCV2) is the main causative agents of post-weaning multi-systemic wasting syndrome leading to significant economic losses in swine population. Although vaccination is the most effective strategy to decrease morbidity and mortality rate, the virus persist in immunized populations and causes subclinical infections with symptoms similar to porcine circovirus diseases. To develop new effective vaccine against PCV2 infection, we prepared chimeric complex VP1-PCV2bCap containing PCV2bCap protein fused with mouse polyomavirus (MPyV). Complex was expressed in baculovirus expression system and purified by nickel agarose. From eluate containing large aggregates of purified proteins, fraction containing pantameres of VP1-PCV2bCap protein was separated by Asymmetrical flow field-flow fractionation and fractions were analyzed by electron microscopy with nano-gold labeling of PCV2Cap portion of the fused protein. 6 weeks old BALB/c mice were subcutaneously vaccinated by 50ug of purified chimeric protein alone or in combination with incomplete Freund adjuvants (50%) or Emulsigen (10%). Immunity response was monitored by indirect ELISA. After the first immunization strong immunity response that gradually increased after the second and third immunization was detected in all mice immunized by chimeric protein. The best immunity response was detected in mice immunized by vaccine with Emulsigen (10%) as adjuvant. Purified chimeric protein VP1-PCV2bCap has a high potential as a DIVA (differentiating infected form vaccinated animals) vaccine against PCV2b infections in pigs. The biggest benefit of this type of vaccine is possibility to distinguish between naturally infected and vaccinated animals

Molecular epidemiology and genetic variation analyses of porcine circovirus type 2 isolated from Yunnan Province in China from 2016-2019

BackgroundPorcine circovirus type 2 (PCV2) is the causative agent of porcine circovirus-associated disease (PCVAD). Its prevalence in swine herds was first reported in China in 2000. PCV2 infection causes immunosuppression that leads to multiple diseases, causing serious economic problems for the swine industry in China. Since information on the genetic variation of PCV2 in Yunnan province is limited, this study aims to investigate the molecular epidemiological and evolutionary characteristics of PCV2 from 2016 to 2019.MethodsA total of 279 clinical samples were collected from different regions of Yunnan between 2016 to 2019, and PCV2 was detected by PCR. We then amplified full genomes from the positive samples, and the sequences were analysed for homology and genetic evolution.ResultsOverall, 60.93% (170/279) of the screened swine herd samples were positive for PCV2. We sequenced 15 Yunnan province PCV2 strains from positive samples. Analyses of the complete genomes and Cap genes led to the classification of the 15 Yunnan PCV2 strains into PCV2a (2 of 15), PCV2b (1of 15) and PCV2d (12 of 15). All strains shared 94.3–99.9% of their identities with the nucleotide sequences of complete genomes in this study and shared 94.2–99.9% identity with the reference sequences. All strains share 89.4–100% and 86.8–100% of their identities with the nucleotide and amino acid (aa) sequences of Cap, respectively.ConclusionsThe results of this study provide evidence that PCV2a, PCV2b and PCV2d genotypes coexisted in Yunnan Province from 2016 to 2019, and the priority prevalence genotype was PCV2d. The data provide evidence for the increased genetic diversity and insights into the molecular epidemiology of PCV2. This study also provides basic data for the Yunnan province PCV2 molecular epidemiological survey and accumulates effective materials for the development of PCV2 vaccines.

Porcine circovirus type 2a or 2b based experimental vaccines provide protection against PCV2d/porcine parvovirus 2 co-challenge

With the discovery of Porcine circovirus type 2d (PCV2d) in the USA in 2012 and subsequent genotype shift from the previously predominant PCV2b to PCV2d in the face of widespread PCV2a vaccination, concerns over PCV2 vaccine efficacy were raised. The objective of this study was to evaluate the efficacy of two similarly produced PCV2 vaccines, one containing the PCV2a capsid and the other one containing the PCV2b capsid, in the conventional pig model against PCV2d/porcine parvovirus 2 (PPV2) co-challenge. A co-challenge was added since there is evidence that PPV2 may exacerbate PCV2 infection and since PCV2 only rarely causes disease in experimentally infected pigs, hence vaccine efficacy can be difficult to assess. In brief, sixty 3-week-old-pigs from a PCV2 seropositive farm without evidence of active virus replication (no PCV2 viremia, low antibody titers with no evidence of increase after two consecutive bleedings) were blocked by PCV2 antibody titer and then randomly divided into three groups with 20 pigs each, a non-vaccinated group (challenge control), a PCV2a vaccinated group (VAC2a) and a PCV2b vaccinated group (VAC2b). Vaccinations were done at 4 and again at 6 weeks of age. At 8 weeks of age, all pigs were challenged with a PCV2d strain via intranasal and intramuscular routes of inoculation followed by intramuscular administration of PPV2 one day later. PCV2 vaccination, regardless of PCV2 genotype, resulted in significantly higher humoral and cellular immunity compared to non-vaccinated challenge control pigs as evidenced by increased numbers of interferon (IFN) γ secreting cells after PCV2d stimulation of peripheral blood mononuclear cells collected prior to challenge. Furthermore, PCV2a and PCV2b vaccinations both reduced PCV2d viremia and PCV2-associated pathological lesions. Under the study conditions, the PCV2a and PCV2b vaccine preparations each induced immune responses and clinical protection against a heterologous PCV2d/PPV2 co-challenge.

Protective humoral immunity in guinea pigs induced by PCV2 virus-like particles displaying the B cell linear epitope (228QQITDA233) of PPV1

Although PCV2 infections generally cause mild disease in pigs, concurrent co-infections with other pathogens can damage the immune system and cause more severe diseases, collectively termed porcine circovirus associated diseases (PCVAD). Involvement of porcine parvovirus (PPV, a common cause of reproductive failure in naïve dams) in PCVAD caused by PCV2, has been reported. As this co-infection can be difficult to eliminate, there is a critical need to develop an effective vaccine to protect against PPV or synergistic effects of PCV2 and PPV under field conditions. In this study, we designed chimeric PCV2 virus-like particles (cVLPs) displaying a B-cell epitope derived from PPV1 structural protein around the surface of the 2-fold axes of PCV2 VLPs, based on 3D-structure analysis of the PCV2 capsid. The cVLPs were successfully prepared, verified by transmission electron microscopy and chromatography, with robust antibody titers against PCV2 and PPV1 produced in mice and guinea pigs. In addition, in guinea pigs challenged with 106 TCID50 PCV2, cVLPs conferred more effective immune protection (based on viral load) than a commercial PCV2 vaccine. Finally, antibody responses and immune protection against PPV were also evaluated. In guinea pigs vaccinated with cVLPs, although PPV antibodies detected by a hemagglutination inhibition (HI) assay appeared later after vaccination in the PCV2 cVLPs group than in the commercial PPV vaccine group, there were fewer PPV genomic DNA copies in the PCV2 cVLPs group than in a PBS group. In conclusion, guinea pigs vaccinated with cVLPs developed effective protective immunity against PCV2 challenge, with some protective immunity against PPV. This study provided valuable research data to pursue molecular design of chimeric epitopes PCV2 VLPs.

First detection and phylogenetic analysis of porcine circovirus type 2 in raccoon dogs

BackgroundPorcine circovirus type 2 (PCV2) is a major emerging virus of porcine circovirus-associated disease (PCVAD), which has brought huge economic losses to the global pig industry. Pigs are well known as the natural reservoir of PCV2. Recently, many researchers have revealed PCV2 could infect many other mammals like mice, calves, minks, dogs and goats. In 2018, our laboratory has admitted six cases of raccoon dogs from Qinhuangdao city of China, which were characterized by inappetence, lethargy, depression, abortion, and sterility.ResultsAt last, six raccoon dog-origin PCV2 strains were isolated in this study. Pairwise-sequence comparisons demonstrated that the six raccoon dog-origin PCV2 strains shared a nucleotide similarity of 92.1–99.8% among 40 PCV2 representative strains. Phylogenetic analysis indicated these PCV2 isolates belonged to Chinese epidemic genotypes PCV2b and PCV2d. And aborted or sterile symptom was significantly associated with PCV2 infection in raccoon dogs by the chi-square test (χ2 = 87.3, p < 0.001). The retrospective study revealed that raccoon dog-origin PCV2 strains shared 100% sequence similarity with the PCV2 stains isolated from pig farms around these raccoon dog farms, respectively.ConclusionIn this study, the first supported evidence of PCV2 prevalence in raccoon dog farms of China was documented. PCV2 may be one of the most significant causative agents resulting in the reproductive failure of farmed raccoon dogs, implying that PCV2 could transmit from pigs to raccoon dogs. That indicated that PCV2 cross-species transmission will be a serious threat to China’s fur animal farming industry.

A radical form of nitric oxide inhibits porcine circovirus type 2 replication in vitro

BackgroundPorcine circovirus type 2 (PCV2) is the causal agent of postweaning multisystemic wasting syndrome (PMWS), causing large economical losses of the global swine industry. Nitric oxide (NO), as an important signaling molecule, has antiviral activity on some viruses. To date, there is little information on the role of NO during PCV2 infection.ResultsWe used indirect fluorescence assay (IFA), TCID50, real-time RT-qPCR and western blot assay to reveal the role of NO in restricting PCV2 replication. PCV2 replication was inhibited by a form of NO, NO•, whereas PCV2 was not susceptible to another form of NO, NO+.ConclusionOur findings indicate that the form of NO• has a potential role in the fight against PCV2 infection.

Seroprevalence of porcine circovirus type 2 infection in the area of Vojvodina

The aim of this research was to determine seroprevalence of infection caused by PCV2 in different categories of pigs on the territory of Vojvodina. The research was conducted on 6 pig farms placed in different locations on the territory of Vojvodina (two farms from the territory of Backa, Banat and Srem, respectively), with the capacity from 500 to 2,500 sows with intensive management, closed type. Blood samples from brachioce?phalic plexus were collected from 540 pigs in three different categories, in suckling piglets between 3 and 4 weeks of age, in weaned piglets from 8 to 9 weeks of age, and in fattening pigs between 20 and 22 weeks of age. Specific anti-PCV2 antibodies are determined by using indirect ELISA. Based on our results, we conclude that PCV2 infections are widely spread on the territory of Vojvodina, with total seroprevalence of 74.8%. The highest value of seroprevalence is determined in category of fattening pigs 86%, in suckling piglets this was 74.5%, while in weaned piglets seroprevalence was 65.3%.

Dissecting clinical outcome of porcine circovirus type 2 with in vivo derived transcriptomic signatures of host tissue responses

BackgroundPorcine Circovirus Type 2 (PCV2) is a pathogen that has the ability to cause often devastating disease manifestations in pig populations with major economic implications. How PCV2 establishes subclinical persistence and why certain individuals progress to lethal lymphoid depletion remain to be elucidated.ResultsHere we present PorSignDB, a gene signature database describing in vivo porcine tissue physiology that we generated from a large compendium of in vivo transcriptional profiles and that we subsequently leveraged for deciphering the distinct physiological states underlying PCV2-affected lymph nodes. This systems genomics approach indicated that subclinical PCV2 infections suppress a myeloid leukocyte mediated immune response. However, in contrast an inflammatory myeloid cell activation is promoted in PCV2 patients with clinical manifestations. Functional genomics further uncovered STAT3 as a druggable PCV2 host factor candidate. Moreover, IL-2 supplementation of primary lymphocytes enabled ex vivo study of PCV2 replication in its target cell, the lymphoblast.ConclusionOur systematic dissection of the mechanistic basis of PCV2 reveals that subclinical and clinical PCV2 display two diametrically opposed immunotranscriptomic recalibrations that represent distinct physiological states in vivo, which suggests a paradigm shift in this field. Finally, our PorSignDB signature database is publicly available as a community resource (http://www.vetvirology.ugent.be/PorSignDB/, included in Gene Sets from Community Contributors http://software.broadinstitute.org/gsea/msigdb/contributed_genesets.jsp) and provides systems biologists with a valuable tool for catalyzing studies of human and veterinary disease. Finally, a primary porcine lymphoblast cell culture system paves the way for unraveling the impact of host genetics on PCV2 replication.

Structural-functional characteristics of thymus in piglets with signs of latent and subclinic PCV2-infection

Circovirus type II disease is an infectious disease with significant economic impacts on the pig industry, which has become global in the pig population (PCVD). The most common form of PCV2-infection has long been considered a PCV2-systemic disease, but recently latent and subclinical forms of PCV2-infection have become of particular importance. The main “target” for PCV-2 is the immune system, and the multiplication of PCV-2 in the cells of the immune system leads to their death and development of immunodeficiency state. The immune system, together with the nervous and endocrine systems, plays an important role in maintaining the body's homeostasis and ensures its adaptation to the effects of unfavorable environmental factors. The organs of the immune system of pigs include bone organs, thymus, spleen, lymph nodes, as well as diffuse and aggregated lymphatic formations of the walls of the digestive canal and respiratory tract. Thymus is the central organ of lymphocytopoiesis and imunopoiesis, in it occurs differentiation of all cellular forms of mature T-lymphocytes in the pre-T-lymphocyte stage, which migrate from the bone marrow to the bloodstream. The growth and development of the thymus, as well as the bone organs, is in direct interaction with the growth and development of the spleen, lymph nodes and is reflected in the structure of the interstitial connective tissue and lymphoid parenchyma, and the question of the structural and functional characteristics of the thymus piglets with signs of latent and subclinical PCV-2 infection in accessible the scientific literature is not sufficiently elucidated and requires a detailed study. Purpose: determination of the structural and functional characteristics of the thymus of piglets with signs of latent and subclinical PCV-2-infection. The work was carried out in pig farms of Ukraine, with intensive technology of pig rearing, at the department of normal and pathological anatomy of agricultural animals and the Scientific and Research Center for Biosafety and Environmental Control of the Agroindustrial Complex Resources of Dnipro State Agrarian and Economic University. To determine the structural and functional characteristics of the thymus of piglets at different forms of the of PCV-2-infection, monitoring studies of blood sera were carried out by quantitative PCR-analysis. A total of 275 samples of blood serum from piglets of 5–16 weeks of age were investigated. For pathomorphological studies with the method of acute bleeding, animals with signs of latent PCV2-infection (in 1 cm3 of blood serum of 103–104 copies of the genome of PCV-2 equivalents) were slaughtered and animals with signs of subclinical PCV2 infection (in 1 cm3 of blood serum 105–106 copies of the gene equivalents of PCV-2) 6 heads. By anatomical preparation, the thymus was removed, fixed in a 10% solution of neutral formalin, poured into paraffin, sections were stained with hematoxylin and eosin and azur II eosin according to generally accepted techniques. In the paraffin sections, the presence of PCV-2 antigen (immunohistochemical staining) was also determined. In the lymphoid parenchyma of the thymus, the percentage ratio between individual cells (large, medium, small lymphocytes, plasmocytes and reticular cells), macrophages and other cells was determined. Cell counting of parenchyma was performed on dasgs stained with azur II eosin at an increase of 10 x 100 under the immersion for every 100 cells on ten preparations in 20 fields of vision for each individual group of animals. Based on the data obtained, the average percent content of each cell type was determined. The author, on the basis of pathohistological and immunohistochemical studies, determined the structural and functional characteristics of the thymus of piglets with signs of latent and subclinical PCV2-infection. It has been established that in piglets with signs of subclinical and latent PCV2-infection, pathogistological changes of the thymus do not differ significantly from the corresponding thymus parameters of clinically healthy animals free from PCV-2. At the same time, there is a tendency to decrease the number of lymphoid cells, mainly immunoblasts and lymphocytes, and, accordingly, the area of the particle cortex, which is more pronounced in thymus of pigs with signs of subclinical PCV2-infection.

PCV2 infection activates the cGAS/STING signaling pathway to promote IFN-β production and viral replication in PK-15 cells

PCV2 is a single-stranded DNA virus that we previously found to induce IFN-β production via RIG-I and MDA-5. cGAS is known to be the most important DNA sensor for the recognition of cytosolic DNA; however, it remains unclear whether the interferon production induced by PCV2 is associated with cGAS. In the present study, PCV2 infection was found to increase the level of cGAS and STING expression, promote the release of cyclic dinucleotide cGAMP, and induce STING dimerization and translocation into the nucleus of PK-15 cells. These findings indicate that PCV2 infection activates both cGAS and STING. Furthermore, the knockdown of cGAS and STING decreased both the mRNA expression and promoter activity of IFN-β, demonstrating that the cGAS/STING signaling pathway contributes to the production of IFN-β. In addition, a knockdown of cGAS and STING also decreased the PCV2 viral load and infectivity. Therefore, PCV2 infection activates the cGAS/STING signaling pathway to induce IFN-β production and the knockdown of cGAS and STING decreases viral replication in PK-15 cells. These results provide further insight into the relationship between PCV2 and host innate immunity.