Porcine Circovirus Type 2 Virus Research Articles

Evaluation of cross-immunity among major porcine circovirus type 2 genotypes by infection with PCV2b and PCV2d circulating strains

There are three main genotypes of porcine circovirus type 2 (PCV2), namely PCV2a, PCV2b and PCV2d, of which PCV2b and PCV2d are currently the most common. There are antigenic differences between these different genotypes. To explore the effect of PCV2 antigen differences on the immune protection provided by vaccines, a cross-immune protection test was carried out in pigs. Three genotype strains, PCV2a-CL, PCV2b-MDJ and PCV2d-LNHC, were inactivated and emulsified to prepare inactivated vaccines to immunize pigs, who were then challenged with the circulating strains PCV2b-BY and PCV2d-LNHC. Immunoperoxidase monolayer assays (IPMAs) and micro-neutralization assays were used to detect antibodies against the three different genotypes of PCV2. The results showed that the three genotype vaccines induced pigs to produce antibodies against the same and different genotypes of PCV2, but the levels of IPMA and neutralizing antibodies against the same genotype were higher than those against different genotypes. Quantitative Polymerase Chain Reaction (qPCR), virus titration and immunohistochemistry were used to detect PCV2 genomic DNA, live virus and antigen, respectively, in inguinal lymph nodes of experimental pigs. Following challenge with the PCV2b-BY strain, the viral DNA load in the inguinal lymph nodes of pigs immunized with the three genotype vaccines was reduced by more than 99 % compared to the unimmunized group. Following challenge with the PCV2d-LNHC strain, the viral DNA loads in the inguinal lymph nodes of pigs immunized with PCV2a, PCV2b and PCV2d genotype vaccines were reduced by 93.8 %, 99.8 % and 98.3 %, respectively, compared to unimmunized controls. In addition, neither live PCV2 virus nor antigen were detected in the inguinal lymph nodes of pigs immunized with any of the genotype vaccines (0/18), but both were detected in the lymph nodes of experimental pigs in the unimmunized control group (6/6). These findings suggest that, although the antigenic differences of the three genotype strains induce significant differences in antibody levels, they seem to have little effect on cross-protection between different genotypes.

Regulatory effect of Panax notoginseng saponins on the oxidative stress and histone acetylation induced by porcine circovirus type 2.

Porcine circovirus type 2 (PCV2) exists widely in swine populations worldwide, and healthy PCV2 virus carriers have enhanced the severity of the infection, which is becoming more difficult to control. This study investigated the regulatory effect of Panax notoginseng saponins (PNS) on the oxidative stress and histone acetylation modification induced by PCV2 in vitro and in mice. In vitro, PNS significantly increased the scavenging capacities of superoxide anion radicals (O2•-) and hydroxyl radicals (•OH) and reduced the content of hydrogen peroxide (H2O2) induced by PCV2 in porcine alveolar macrophages (3D4/2). In addition, PNS decreased the protein expression level of histone H4 acetylation (Ac-H4) by increasing the activity of histone deacetylase (HDAC) in PCV2-infected 3D4/2 cells. In vivo, PNS enhanced the scavenging capacities of •OH and O2•− and reduced the content of H2O2 in the spleens of PCV2-infected mice. PNS also reduced the protein expression level of histone H3 acetylation (Ac-H3) by reducing the activity of histone acetylase (HAT) and increasing the activity of HDAC in the spleens of PCV2-infected mice. PCV2 infection activated oxidative stress and histone acetylation in vitro and in mice, but PNS ameliorated this oxidative stress. The research can provide experimental basis for exploring the antioxidant effect and the regulation of histone acetylation of PNS on PCV2-infected 3D4/2 cells and mice in vitro and in vivo, and provide new ideas for the treatment of PCV2 infection.

Genetic diversity of Indian porcine circovirus type 2 (PCV2) isolates (2006-2018)

Porcine circovirus type 2 (PCV2) is an emerging viral infection in swine population and results in severe economic loss to piggeries worldwide. The present study was conducted to explore the genetic diversity of PCV2 circulating in swine population of India from 2006–2018. A total of 74 heart, lungs, spleen and lymph nodes collected from different regions in Uttar Pradesh, India were subjected to molecular analysis. For studying genetic diversity, the complete nucleotide and ORF2 sequences of 95 PCV2 including 43 nucleotide sequences from India were used. DNA was extracted from samples and positive samples were subjected to full genome sequencing and phylogenetic analysis was done by maximum likelihood method. Phylogenetic analysis of 40 Indian PCV2 genomes downloaded from GenBank along with three new isolates from the current study based on the complete genome and cap gene together with nucleotide sequences of PCV2 isolates from different countries results in a tree in which Indian isolates clustered in 4 different branches includes PCV2b-1C, PCV2a-2D, PCV 2b-1A/B, PCV 2d-2 recombinant group and two new cluster in which one clustered along with unclassified PCV2 viruses from Indonesia and Croatia. The comparison of ORF2 gene among Indian isolates revealed nucleotide identity ranging from 88.6% to 99.6%, indicating the genetic diversity of PCV2 strains circulating in Indian pig. The present work reports for the first time in India the PCV2-1A/B cluster of 2b genotype and all the Indian isolates available in India from 2006–2018 were used in this analysis.

NAP1L4 inhibits porcine circovirus type 2 replication via IFN-β signaling pathway

Porcine circovirus type 2 (PCV2) capsid protein (Cap) was previously reported to interact with nucleosome assembly protein 1-like 4 (NAP1L4). The biological function of Cap-NAP1L4 interaction is unknown. Here, we demonstrated that PCV2 Cap could directly interact with NAP1L4, which the amino acid residues 124–279 of NAP1L4 were responsible for the interaction. Furthermore, over-expression of NAP1L4 down-regulated the expression of PCV2 Cap and Rep. DNA copies and virus titers were also decreased in NAP1L4 over-expressed PK15 cells. While, knockout of NAP1L4 through CRISPR/Cas9 technology in PK15 cells could up-regulate the mRNA and protein levels of PCV2 Cap and Rep. PCV2 genomic DNA copies and virus titers were also increased in NAP1L4-knockdown/-knockout PK15 cells compared with wild type PK15 cells. In addition, NAP1L4 depletion was demonstrated to facilitate cytosolic carboxypeptidase-like protein 5 (CCP5) and cytosolic carboxypeptidase 6 (CCP6) expression, which could activate cGAS to promote IFN-β production. Indeed, knockout of NAP1L4 was also confirmed to increase IFN-β expression. And IFN-β stimulation could promote PCV2 replication in PK15 cells. Taken together, our findings suggest that NAP1L4 interacts with PCV2 Cap and inhibits PCV2 replication through regulating IFN-β production. Our study provides theoretical basis for further study of PCV2.

The Carboxyl Terminus of the Porcine Circovirus Type 2 Capsid Protein Is Critical to Virus-Like Particle Assembly, Cell Entry, and Propagation.

The capsid protein (Cap) is the sole structural protein and the main antigen of porcine circovirus type 2 (PCV2). Structural loops of the Cap play crucial roles in viral genome packaging, capsid assembly, and virus-host interactions. Although the molecular mechanisms are yet unknown, the carboxyl terminus (CT) of the PCV2 Cap is known to play critical roles in the evolution, pathogenesis, and proliferation of this virus. In this study, we investigated functions of CT. Removal of this loop leads to abrogation of the in vitro Cap self-assembly into virus-like particles (VLPs). Likewise, the mutated virus resists rescue from PK15 cell culture. A conserved PXXP motif in the CT is dispensable for VLP assembly and subsequent cell entry. However, its removal leads to the subsequent failure of virus rescued from PK15 cells. Furthermore, substituting either the PCV1 counterpart or an AXXA for the PXXP motif still supports virus rescue from cell culture but results in a dramatic decrease in viral titers compared with wild type. In particular, a strictly conserved residue (227K) in the CT is essential for VLP entry into PK15 cells, and its mutation to alanine greatly attenuates cell entry of the VLPs, supporting a mechanism for the failure to rescue a mutated PCV2 infectious DNA clone (K227A) from PK15 cell culture. These results suggest the CT of the PCV2 Cap plays critical roles in virus assembly, viral-host cell interaction(s), and virus propagation in vitroIMPORTANCE The carboxyl terminus (CT) of porcine circovirus type 2 (PCV2) capsid protein (Cap) was previously reported to be associated with immunorecognition, alterations of viral titer in swine sera, and pathogenicity. However, the molecular mechanisms underlying these effects remain unknown. In this study, roles of the critical residues and motifs of the CT are investigated with respect to virus-like particle (VLP) assembly, cell entry, and viral proliferation. The results revealed that the positively charged 227K of the CT is essential for both cell entry of PCV2 VLPs and virus proliferation. Our findings, therefore, suggest that the CT should be considered one of the key epitopes, recognized by neutralizing antibodies, for vaccine design and a target for drug development to prevent PCV2-associated diseases (PCVADs). Furthermore, it is important to respect the function of 227K for its role in cell entry if using either PCV2 VLPs for nanoscale DNA/drug cell delivery or using PCV2 VLPs to display a variety of foreign epitopes for immunization.

A Heterologous Viral Protein Scaffold for Chimeric Antigen Design: An Example PCV2 Virus Vaccine Candidate.

Recombinant vaccines have low-cost manufacturing, regulatory requirements, and reduced side effects compared to attenuated or inactivated vaccines. In the porcine industry, post-weaning multisystemic disease syndrome generates economic losses, characterized by progressive weight loss and weakness in piglets, and it is caused by porcine circovirus type 2 (PCV2). We designed a chimeric antigen (Qm1) to assemble the main exposed epitopes of the Cap-PCV2 protein on the capsid protein of the tobacco necrosis virus (TNV). This design was based on the Cap-N-terminal of an isolated PCV2 virus obtained in Chile. The virus was characterized, and the sequence was clustered within the PCV2 genotype b clade. This chimeric protein was expressed as inclusion bodies in both monomeric and multimeric forms, suggesting a high-molecular-weight aggregate formation. Pigs immunized with Qm1 elicited a strong and specific antibody response, which reduced the viral loads after the PCV2 challenge. In conclusion, the implemented design allowed for the generation of an effective vaccine candidate. Our proposal could be used to express the domains or fragments of antigenic proteins, whose structural complexity does not allow for low-cost production in Escherichia coli. Hence, other antigen domains could be integrated into the TNV backbone for suitable antigenicity and immunogenicity. This work represents new biotechnological strategies, with a reduction in the costs associated with vaccine development.

T cell epitope content comparison (EpiCC) analysis demonstrates a bivalent PCV2 vaccine has greater T cell epitope overlap with field strains than monovalent PCV2 vaccines

Porcine circovirus type 2 (PCV2) has one of the highest evolutionary rates among DNA viruses. Traditionally, PCV2 vaccines have been based on the 2a genotype as this was the first genotype discovered. Today, eight genotypes of PCV2 viruses have been identified, and, taken together with the rapid evolutionary rate, propensity to recombine, and high rate of vaccination, further variation in PCV2 is expected. For these reasons, there is a growing genetic gap between available vaccines and field strains. When selecting vaccines, it is important to consider vaccines that contain T cell epitopes that are well-matched to the circulating strains. To quantify the relatedness between PCV2 vaccines and field strains, we predicted and compared their T cell epitope content and calculated Epitope Content Comparison (EpiCC) scores using established in silico tools. T cell epitopes predicted to bind common class I and class II swine leukocyte antigen (SLA) alleles were identified from two major structural proteins, the capsid (encoded by ORF2) and the replicase (encoded by ORF1). The T cell epitope content of three commercial PCV2a-based vaccines (a baculovirus expressed PCV2a ORF2 [VacAlt], a PCV1-PCV2a chimeric virus vaccine [VacA] and a combination cPCV2a-cPCV2b chimeric virus vaccine [VacAB]) and an experimental PCV2b ORF2-based chimeric virus vaccine [VacB] (Table 1), were compared to that of 161 PCV2 field strains (representing genotypes a-f).The T cell epitope content and conservation between vaccine and field strains varied. While all vaccine strains provided broad coverage of the field strains including heterologous genotypes, none of the vaccines covered all the putative T cell epitopes identified in the field strains. PCV2a-based vaccine strains generally scored higher in terms of conserved epitope content against PCV2a field isolates but were not identical. The PCV2b-based vaccine strain had higher scores against PCV2b and PCV2d field strains. The combination PCV2a-PCV2b vaccine (VacAB) had, on average, the highest EpiCC score. PCV2 continues to evolve and EpiCC analysis provides a new tool to assess the possible impact of virus genetic divergence on T cell epitope coverage of vaccine strains. Given that multiple genotypes are currently found and may co-exist on farms, this analysis suggests that a combination of PCV2a and PCV2b vaccine strains may be required to provide optimal coverage of current and future field isolates.

Efficient production of porcine circovirus virus-like particles using the nonconventional yeast Kluyveromyces marxianus

Porcine circovirus type 2 (PCV2) is a ubiquitous virus with high pathogenicity closely associated with the postweaning multisystemic wasting syndrome (PMWS) and porcine circovirus diseases (PCVDs), which caused significant economic losses in the swine industry worldwide every year. The PCV2 virus-like particles (VLPs) are a powerful subunit vaccine that can elicit high immune response due to its native PCV2 virus morphology. The baculovirus expression system is the widely used platform for producing commercial PCV2 VLP vaccines, but its yield and cost limited the development of low-cost vaccines for veterinary applications. Here, we applied a nonconventional yeast Kluyveromyces marxianus to enhance the production of PCV2 VLPs. After codon optimization, the PCV2 Cap protein was expressed in K. marxianus and assemble spontaneously into VLPs. Using a chemically defined medium, we achieved approximately 1.91g/L of PCV2 VLP antigen in a 5-L bioreactor after high cell density fermentation for 72h. That yield greatly exceeded to recently reported PCV2 VLPs obtained by baculovirus-insect cell, Escherichia coli and Pichia pastoris. By the means of two-step chromatography, 652.8mg of PCV2 VLP antigen was obtained from 1L of the recombinant K. marxianus cell culture. The PCV2 VLPs induced high level of anti-PCV2 IgG antibody in mice serums and decreased the virus titers in both livers and spleens of the challenged mice. These results illustrated that K. marxianus is a powerful yeast for cost-effective production of PCV2 VLP vaccines.

Production of Escherichia coli-based virus-like particle vaccine against porcine circovirus type 2 challenge in piglets: Structure characterization and protective efficacy validation

We report the strategies leading to the large-production of soluble non-tag full-length porcine circovirus type 2 (PCV2) Cap protein in Escherichia coli. Under neutral pH condition, the purified recombinant Cap protein derived from E. coli expression self-assembles into homogenous round virus-like particle at the similar size of that of the intact PCV2 virus, which is further characterized by Cryo-EM single particle structure determined at 4.5Å. The engineered PCV2 rCap VLP was tested as a subunit vaccine for the protective efficacy against PCV2 challenge on 3-week old piglets. Similar to commercial available PCV2 vaccine, the Cap VLP-immunized piglets developed specific antibody-mediated response and were protected from the virulent SH PCV2 strain challenge. Hence, the production of E. coli based PCV2Cap-VLP could be applied as a cost-friendly and effective subunit vaccine to control PCV2 spreading in developing countries.

Effect of dietary selenium yeast supplementation on porcine circovirus type 2 (PCV2) infections in mice.

The present study was performed to determine the protective role of dietary selenium (Se) yeast supplementation in porcine circovirus type 2 (PCV2) infected mice. Forty-eight Kun Ming female mice were randomly assigned to Se yeast group (0.3%Se +basal diet, n = 24) and control group (basal diet, n = 24). After 3 days of adaptive feeding and 15 days treatment with the experimental feed, mice were challenged by intraperitioneal injection of PCV2 at the dosage of 2000 TCID50 (50% tissue culture infection dose, TCID50). Serum total superoxide dismutase (SOD) activity, malondialdehyde (MDA) level, tumor necrosis factor alpha (TNF-α), C-reactive protein (CRP) and interleukin-1 beta (IL-1β) levels were measured at 5, 10, 15, 20 days post infection (dpi). The PCV2 virus load in the liver, spleen and lung, and the microscopic lesions in the liver, spleen and lung also were determined on 5, 10, 15, and 20 dpi. Dietary Se yeast supplementation decreased (Pμ0.05) the serum levels of TNF-α, but had no significant effect on the activity of SOD and the levels of MDA, CRP and IL-1β between experimental and control groups. Dietary Se yeast supplementation had little effect on the PCV2 virus load in the liver, spleen and lung. However, mice in the selenium yeast group showed a significant decrease in microscopic lesion scores in the lung and spleen compared with those in the control group (Pμ0.05). These data indicate Se yeast attenuated the PCV2 infection through altering the systemic inflammation and maintaining the normal organ morphology.

Genetic characterisation and phylogenetic analysis of PCV2 isolates from India: Indications for emergence of natural inter-genotypic recombinants

Porcine circovirus type 2 (PCV2), the necessary agent in pathogenesis of porcine circovirus diseases (PCVDs), has a worldwide distribution and is considered as one of the most important emerging viral pathogens of economic importance. PCV2 has been divided into four major genotypes namely PCV2a with five clusters or subtypes (2A–2E), PCV2b with three clusters (1A–1C), PCV2c and PCV2d, based on capsid (cap) gene analysis. PCV2 genome is rapidly evolving through events of recombination and mutation. Though, PCV2a was the predominant genotype initially, PCV2b shared majority of PCV2 sequences submitted to GenBank since 2003. In India, data regarding molecular characterisation of PCV2 is scant or absent. In the present study, we thoroughly analysed genetic heterogeneity of PCV2 strains circulating in Indian pig population. The results revealed that pigs in this region harboured PCV2 viruses of different genotypes including PCV2a–2D, PCV2b–1C and PCV2d. More interestingly, two isolates (PCV2Izn-89-13 and PCV2Izn-218-13) were classified as recombinant strains. Further detailed analysis suggested that these strains evolved from inter-genotypic recombination between PCV2a–2C and PCV2b–1C genotypes within cap gene. This study reports for the first time, the emergence of recombinant PCV2 strains in the Indian pig population.

Immune responses of piglets immunized by a recombinant plasmid containing porcine circovirus type 2 and porcine interleukin-18 genes.

In this study, two recombinant plasmids containing the ORF2 gene of porcine circovirus type 2 (PCV2) with or without porcine interleukin-18 (IL-18) were constructed and evaluated for their ability to protect piglets against PCV2 challenge. Transient expression of the plasmids in PK-15 cells could be detected using Western blot. Piglets were given two intramuscular immunizations 3 weeks apart and were challenged with a virulent Wuzhi strain of PCV2 at 42 days after the initial immunization. All animals vaccinated with pBudCE4.1-ORF2 or with pBudCE4.1-ORF2/IL18 developed PCV2-specific antibody and T-lymphocyte proliferative responses. The levels of T-lymphocyte proliferation in piglets immunized with pBudCE4.1-ORF2/IL18 were significantly higher than in those immunized with pBudCE4.1-ORF2, and pBudCE4.1-ORF2/IL18 stimulated a significantly increased production of IFN-γ and IL-2. Furthermore, PCV2 challenge experiments showed that the DNA vaccine-immunized groups can partially prevent PCV2 viremia and significantly reduce the amount of PCV2 virus in the lymphoid tissues, and the piglets immunized by pBudCE4.1-ORF2/IL18 exhibit a marked inhibition of PCV2 replication compared to the pBudCE4.1-ORF2 group. These data demonstrate that the plasmid pBudCE4.1-ORF2/IL18 may be an effective approach for increasing PCV2 DNA vaccine immunogenicity.

The presence of six potentially pathogenic viruses in pigs suffering from post-weaning multisystemic wasting syndrome.

BackgroundPorcine circovirus type 2 (PCV2) is an etiological agent of porcine circovirus diseases (PCVDs). Post-weaning multisystemic wasting syndrome (PMWS) as the most important PCVD is considered a multifactorial disease. It was demonstrated that not only PCV2 but several viruses are associated with PMWS. Studies of viral co-infections in PMWS pigs led often to controversial results. The aim of this work was to determine the presence of emerging (PRRSV), re-emerging (PTV) and newly-emerging (TTSuV1, TTSuV2, PBoV1) viruses in samples of dead pigs suffering from PMWS. The impact of vaccination against PCV2 and the influence of age on the occurrence of single and multiple viral infections in pigs were also investigated.ResultsViruses were detected by PCR, RT-PCR and real-time PCR in the pooled tissue samples (lymph nodes, liver and spleen) of pigs with PMWS (n = 56) which were divided into three groups: suckling piglets, post-weaning pigs and fattening pigs. In addition, lymph node samples were collected from apparently healthy fattening pigs (n = 59). The effect of vaccination against PCV2 with Ingelvac CircoFlex vaccine was also investigated. Between non-vaccinated pigs, the highest prevalence of individual viruses and multiple viral infections were found in diseased post-weaning and fattening animals with PMWS. Severe clinical disease was observed in swine co-infected with PCV2 and PRRSV. The prevalence of TTSuV1 and TTSuV2 was high in all groups of pigs and did not appear to have a significant effect on the syndrome. Simultaneous infection with TTSuV1 and PBoV1 was frequently confirmed in pigs with PMWS. No healthy pig was found to be infected with PRRSV, PTV or PBoV1. Vaccination against PCV2 did not influence the prevalence of TTSuVs, but significantly protected pigs against multiple viral infections.ConclusionsPost-weaning PMWS pigs were more often co-infected with viral pathogens than suckling or fattening pigs. Co-infection with PRRSV enforces clinical signs of PMWS, the influence of other viral co-infections is not clear. Vaccination against PCV2 significantly reduced viral co-infections in pigs.

Dietary supplementation with proline confers a positive effect in both porcine circovirus-infected pregnant and non-pregnant mice

Porcine circovirus type 2 (PCV2) is associated with various diseases that impose a significant economic burden on the swine industry. We hypothesised that nutritional supplementation with proline to enhance the immune response might be a useful prophylactic measure against PCV2 infection. To test this hypothesis, in the present study, we measured clinical data, including blood parameters, serum cytokine profile, PCV2 virus load in organs and serum, and microscopic lesions in the lung, liver and spleen, in both PCV2-infected pregnant and non-pregnant mice. Dietary supplementation with proline had no effect (P>0·05) on abortion rates in PCV2-infected pregnant mice, although a numerically lower abortion rate (22·2 v. 44·4%) was observed compared with the control. Dietary supplementation with proline significantly increased serum C-reactive protein levels (P= 0·03) in PCV2-infected pregnant mice, and increased serum TNF-α levels (P= 0·01), leucocytes (P< 0·05), lymphocytes (P< 0·05) and neutrophilic granulocytes (P< 0·05) in PCV2-infected non-pregnant mice. Meanwhile, dietary proline significantly (P< 0·05) decreased the PCV2 virus load in the lung. Furthermore, mice in the dietary proline group showed a significant (P< 0·01) decrease in microscopic lesion scores in the lung, liver and spleen compared with those in the alanine group. Collectively, dietary proline supplementation confers a functional role in PCV2-infected mice.

Comparison of three commercial one-dose porcine circovirus type 2 (PCV2) vaccines on PCV2 shedding in semen from experimentally infected boars

This study compared the effects of 3 different types of commercial PCV2 vaccines on PCV2 virus shedding in the semen from infected boars. Twenty-five non-PCV2 viremic and seronegative boars were randomly divided into five groups: three vaccinated and challenged groups, a non-vaccinated and challenged group, and a negative control group. The number of genomic copies of PCV2 in serum and semen samples was significantly decreased in vaccinated and challenged boars compared to non-vaccinated and challenged boars from 14 to 70 days post-inoculation (dpi). The number of PCV2 genomic copy in the semen correlated with the number of PCV2b genomic copy in the blood in vaccinated and challenged boars (r(2)=0.894-0.926, P<0.01), and non-vaccinated and challenged boars (r(2)=0.903, P<0.01). The vaccination protocol reduced the amount of PCV2 DNA shed in the semen. However, there was a significantly different amount of PCV2 DNA shed in semen among the 3 vaccinated and challenged boar groups.

Glutamine modifies immune responses of mice infected with porcine circovirus type 2

The present study was conducted to evaluate the immune-enhancing effects of dietary L-glutamine supplementation in porcine circovirus type 2 (PCV2)-infected mice, and to examine the clearance effects of glutamine against PCV2 in experimentally infected mice. A total of sixty Kunming female mice were infected with PCV2 at a dose of 100 TCID(50) (50% tissue culture infection dose) by intraperitoneal injection after 2 weeks of dietary L-glutamine supplementation or L-alanine supplementation (as the control (isonitrogenous) group). The measured variables on 3rd, 5th, 7th, 9th and 11th d post-infection (dpi) included: (1) PCV2 virus loaded in the liver, spleen, heart, lung, kidney, ovary and serum was determined by real-time PCR; (2) IL-2, IL-6, IL-10, interferon (IFN)-α, IFN-γ and C-reactive protein levels in serum were measured by ELISA; (3) serum total superoxide dismutase activity was measured spectrophotometrically at 550nm absorbance. Dietary L-glutamine supplementation significantly increased serum IL-2 levels on the 3rd (P<0·01), 5th (P<0·01), 7th (P<0·05) and 9th dpi, significantly (P<0·05) increased serum IL-6 levels on 3rd dpi, significantly (P<0·05) increased serum IFN-g levels on the 9th and 11th dpi and significantly decreased (P<0·01) serum IL-10 levels on the 9th and 11th dpi, compared with those in the control group. Meanwhile, the PCV2 virus genome was detected sporadically throughout the experimental period in both groups. Taken together, the present results suggest that dietary L-glutamine supplementation enhances immune function in PCV2-infected mice.

Efficacy of single dose of an inactivated porcine circovirus type 2 (PCV2) whole-virus vaccine with oil adjuvant in piglets

BackgroundPost-weaning multisystemic wasting syndrome (PMWS) associated with PCV2 is one of the most costly diseases currently faced by the swine industry. The development of effective vaccines against PCV2 infection has been accepted as an important strategy in the prophylaxis of PMWS.MethodsIn the present study, a PK-15 cell-adapted formalin-inactivated prototype vaccine candidate was prepared using a strain of PCV2 from China. Inactivation of the virus was accomplished using a standard formalin inactivation protocol. The protective properties of the inactivated PCV2 vaccine were evaluated in piglets. Ten 28-day-old pigs were randomly assigned to two groups, each with five. Group 1 was vaccinated intramuscularly with the inactivated virus preparation; Group 2 received sterile PBS as a placebo. By 28 days post-vaccination (DPV), Groups 1 and 2 were challenged intranasally and intramuscularly with 5 × 107 TCID50 of a virulent PCV2 isolate.ResultsThe vaccinated pigs seroconverted to PCV2 and had high levels of serum antibodies to PCV2 at 28 days after vaccination, whereas the control pigs remained seronegative. No significant signs of clinical disease were recorded following the challenge with PCV2, but moderate amounts of PCV2 antigen were detected in most lymphoid organs of the control pigs. PCV2 was detected in two out of the five vaccinated pigs. Furthermore, pathological lesions and viremia were milder in the vaccinated group.ConclusionsThe obtained results indicate that the inactivated PCV2 virus vaccine with an oil adjuvant induce an immunological response in pigs that appears to provide protection from infection with PCV2. The vaccine, therefore, may have the potential to serve as a vaccine aimed to protect pigs from developing PMWS.

In Vitro and In Vivo Isolation of a Novel Rearranged Porcine Circovirus Type 2

Here, we present the first report of a novel rearranged porcine circovirus type 2 (PCV2) strain named BIV, isolated from both in vitro and in vivo sources. The complete circular genome of BIV is 896 nucleotides in length. The data will help us to update current knowledge of the replication of PCV2 viruses in cell culture and of their molecular evolution, as well as their diagnosis.

Evaluation of the transmission of porcine circovirus type 2 (PCV-2) genogroups a and b with semen from infected specific-pathogen-free boars

The porcine circovirus type 2 (PCV-2) is associated with several diseases including reproductive failure. This syndrome has been experimentally reproduced twice with two PCV-2 isolates representative of each major PCV-2 genogroup, i.e. PCV-2a and PCV-2b (Cariolet et al., 2002; Rose et al., 2007). In these two previous studies, the sows were infected by intra-uterine inoculation at insemination with 104.3 and 103.18 TCID50 of PCV-2a and PCV-2b, respectively, corresponding to 1.2×1011 and 3×1010 genome copies, respectively.The aim of this present study was to quantify viral shedding in semen from specific-pathogen-free (SPF) boars infected with isolates from the two major PCV-2 genogroups a and b. We studied the transmission of the PCV-2 virus through contaminated semen to SPF sows and their offspring. The four inoculated boars developed sub-clinical PCV-2 infections and PCV-2 genomes were occasionally detected in semen after nasal infection of boars, with up to 1.2×106copies/mL in the sperm-rich fraction. When PCV-2-contaminated semen was inoculated in SPF sows at artificial insemination, the sows and their offspring did not show any signs of PCV-2 infection or PCV-2 antibodies or genomes. In the present study, sows were inoculated with a maximal dose of 1.7×107 viral genome copies, which is lower than the genomic loads (i.e. 1.2×1011 and 3×1010 genome copies) that have been shown to induce reproductive troubles in intra-uterine inoculated sows. Our results together with the previous experiment findings suggest that PCV-2-induced reproductive disorders depend on the infectious dose inoculated to sows by the intra-uterine route.

ORF1 but not ORF2 dependent differences are important for in vitro replication of PCV2 in porcine alveolar macrophages singularly or coinfected with PRRSV

The objective of this study was to investigate cytokine expression and in vitro replication of porcine circovirus type 2 (PCV2) and porcine reproductive and respiratory syndrome virus (PRRSV) in pulmonary alveolar macrophages (PAMs) emphasizing PCV2 open-reading frame (ORF) origin (PCV2a or PCV2b) and PRRSV strain. Chimeric PCV2 viruses composed of different combinations of ORF1 and ORF2 of PCV2a or PCV2b (chimera PCV2a–2b and chimera PCV2b–2a) were constructed and five different PRRSV isolates were utilized: Type 1 (SD 01-08) or type 2 (NC16845b, VR-2332, MN-184, JA-142). PAMs were infected singularly or with combinations of PCV2b, PCV2a, chimera PCV2a–2b, and chimera PCV2b–2a, and one of the five PRRSV isolates. Real-time PCR was used to test PAMs (PCV2 mRNA) and supernatants (PRRSV RNA, PCV2 DNA, PCV2 mRNA) harvested at 24, 48, 72 and 96h post inoculation (hpi). Levels of IFN-γ, TNF-α and IL-10 were determined by quantitative ELISAs. PCV2 replication in PAMs was limited to groups inoculated with PCV2 strains containing ORF1 of PCV2a (PCV2a, chimera PCV2a–2b). Furthermore, in supernatants, PCV2 mRNA was only detected in groups coinfected with PRRSV regardless of strain at 48hpi supporting an enhancing effect of PRRSV on PCV2 infection. Changes in cytokine levels were minimal and associated with PRRSV strain for TNF-α. In summary, in vitro differences in PCV2 replication in PAMs inoculated with different PCV2–PRRSV combinations were independent of PCV2 ORF2 origin with minimal effects of concurrent PRRSV infection perhaps indicating that PCV2-specific changes in ORF1 may be more important than those in ORF2.