Chimeric Porcine Parvovirus VP2 Virus-like Particles with Epitopes of South African Serotype 2 Foot-and-Mouth Disease Virus Elicits Specific Humoral and Cellular Responses in Mice.

Southern Africa Territories 2 (SAT2) foot-and-mouth disease (FMD) has crossed long-standing regional boundaries in recent years and entered the Middle East. However, the existing vaccines offer poor cross-protection against the circulating strains in the field. Therefore, there is an urgent need for an alternative design approach for vaccines in anticipation of a pandemic of SAT2 Foot-and-mouth disease virus (FMDV). The porcine parvovirus (PPV) VP2 protein can embed exogenous epitopes into the four loops on its surface, assemble into virus-like particles (VLPs), and induce antibodies and cytokines to PPV and the exogenous epitope. In this study, chimeric porcine parvovirus VP2 VLPs (chimeric PPV-SAT2-VLPs) expressing the T-and/or B-cell epitopes of the structural protein VP1 of FMDV SAT2 were produced using the recombinant pFastBac™ Dual vector of baculoviruses in Sf9 and HF cells We used the Bac-to-Bac system to construct the recombinant baculoviruses. The VP2-VLP--SAT2 chimeras displayed chimeric T-cell epitope (amino acids 21-40 of VP1) and/or the B-cell epitope (amino acids 135-174) of SAT FMDV VP1 by substitution of the corresponding regions at the N terminus (amino acids 2-23) and/or loop 2 and/or loop 4 of the PPV VP2 protein, respectively. In mice, the chimeric PPV-SAT2-VLPs induced specific antibodies against PPV and the VP1 protein of SAT2 FMDV. The VP2-VLP-SAT2 chimeras induced specific antibodies to PPV and the VP1 protein specific epitopes of FMDV SAT2. In this study, as a proof-of-concept, successfully generated chimeric PPV-VP2 VLPs expressing epitopes of the structural protein VP1 of FMDV SAT2 that has a potential to prevent FMDV SAT2 and PPV infection in pigs.

Viral Fitness and Antigenic Determinants of Porcine Parvovirus at the Amino Acid Level of the Capsid Protein.

Since 2001, strains of porcine parvovirus (PPV), designated 27a-like strains, were observed in Europe, suggesting a predominance of these viruses over older strains. The reasons for the obvious evolutionary advantage are unknown. Here, a series of mutants containing amino acid replacements found in the predominant field strains were generated in a PPV-NADL2 background, and their impact on replication efficiency and antibody binding activity was determined. Some amino acid substitutions observed in the 27a-like strains significantly increased viral fitness and decreased neutralization activity of serum samples raised against commercial vaccines and old virus strains (e.g., NADL2). These mutant viruses and a monoclonal antibody raised against a classical PPV strain defined a 27a-specific neutralizing epitope around amino acid 228 of the capsid protein VP2. Based on the analysis of the mutant viruses, it is hypothesized that the predominant factor for the global spread of the PPV-27a strain substitutions is an increased viral fitness of the 27a-like viruses, possibly supported by partial immune selection. This is reminiscent to the evolution of canine parvovirus and worldwide replacement of the original virus by the so-called new antigenic types. IMPORTANCE Porcine parvovirus is one of the most important causes of reproductive failure in swine. Recently, despite the continuous use of vaccines, "new" strains emerged, leading to the hypothesis that the emergence of new amino acid substitutions could be a viral adaptation to the immune response against the commercial vaccines. Our results indicate the amino acid substitutions observed in the 27a-like strains can modify viral fitness and antigenicity. However, an absolute immune escape was not evident.

Identification of a dominant linear epitope on the VP2 capsid protein of porcine parvovirus and characterization of two monoclonal antibodies with neutralizing abilities.

Porcine parvovirus (PPV) is a major cause of reproductive failure in swine, and has caused huge losses throughout the world. The structural viral protein VP2, which is able to self-assemble into empty capsids, known as virus-like particles (VLPs), is crucial to induce PPV-specific neutralizing antibodies and protective immunity. In this study, twelve monoclonal antibodies (mAbs) against PPV were generated. The mAbs were characterized by indirect enzyme-linked immunosorbent assay (ELISA), western blotting (WB) and virus neutralization (VN) assay. Two mAbs were defined to be able to neutralize the standard PPV 7909 strain. Subsequently, peptide scanning was applied to identify linear epitopes. The peptide, 89ESGVAGQMV97 was defined as a precise linear epitope. Results from structural analysis showed that the epitope was exposed on the virion surface. Multiple sequence alignment analysis indicated that peptide 89ESGVAGQMV97 was not completely conserved, with a higher amino acid mutation rate at 91G, 92V and 93A position. Alanine-scanning mutagenesis further revealed that residues 89E, 90S, 91G, 92V and 94G were the core sites involved in antibody recognition. These findings may facilitate further understanding the function of the VP2 protein and development of diagnostic tools.

The immunogenicity of the virus-like particles derived from the VP2 protein of porcine parvovirus.

Porcine parvovirus (PPV) is a major cause of the syndrome of sow reproductive failure that can cause economic losses. In this study, we developed a subunit vaccine against porcine parvovirus (PPV), composed of virus-like particles (VLPs) derived from a prokaryotic system, and evaluated its potential against PPV infection. The soluble recombinant VP2 protein was expressed in E. coli Transetta(DE3) cells using a pCold II prokaryotic expression vector at a low temperature of 15 °C. After expression and purification, the recombinant VP2 protein was successfully assembled into VLPs with a similar shape of PPV viron and also hemagglutination activity. PPV VLPs formulated in a water-in-oil-in-water adjuvant evoked high hemagglutination inhibition antibody and neutralization antibody titres in both guinea pigs, used as reference model, and target species, pigs. Immunization with VLPs vaccine stimulated high hemagglutination inhibition antibody and neutralization antibody responses in guinea pigs, used as reference, and target species, weaned pigs, and primiparous gilts. PPV VLPs from E. coli yielded complete fetal protection against PPV infection in primiparous gilts immunized with a single-dose vaccine. PPV VLPs inhibited the replication and spread of PPV in primiparous gilts, which was confirmed by the detection of PPV DNA and infectious PPV in nasal and rectal swabs of challenged sows. These results suggest that VLPs-based PPV vaccine is a promising PPV vaccine candidate.

Gilt Vaccination with a Mixed Administration of a PRRS MLV and a PPV1 Subunit Vaccine Protects against Heterologous PRRSV1 Infection and Prevents Detrimental Effects on Piglet Performance.

The efficacy of the combined administration of a porcine reproductive and respiratory syndrome (PRRS) modified live virus (MLV) vaccine and a porcine parvovirus 1 (PPV1) subunit vaccine in gilts was addressed in two experiments. Experiment A aimed to establish a 4-week onset of immunity (OOI). Gilts were randomly distributed in three treatment groups: non-vaccinated control animals (group 1), animals vaccinated with the combined vaccine (group 2), and a third group that consisted of animals vaccinated with the PRRS MLV vaccine alone (group 3). Four weeks after the first vaccination, gilts were challenged with a heterologous PRRS virus 1 (PRRSV1) and euthanized three weeks after. Besides this, experiment B pursued a 17-week duration of immunity (DOI). In this case, gilts were distributed in the same treatment groups, but for the third group, which consisted of non-vaccinated, non-challenged animals were used instead. For the DOI assessment, gilts were artificially inseminated 4 weeks after the first vaccination, challenged at day 90 of gestation, and followed up, together with their offspring, until day 20 post-farrowing. Serology and viremia post-challenge were determined in gilts from both experiments, while farrowing and piglet performance were only evaluated in experiment B. Overall, the combined vaccine helped to protect gilts from viremia post-challenge and, consequently, to prevent PRRS clinical symptoms and diminish the proportion of piglets infected congenitally or early in life. The combined vaccine also elicited a significant improvement in piglet survival rate and growth performance until weaning. The present results reveal efficacy and lack of interference of the mixed use of the tested vaccines against PRRSV1 infection, with at least 4-week OOI and 17-week DOI.

Duration of immunity against heterologous porcine parvovirus 1 challenge in gilts immunized with a novel subunit vaccine based on the viral protein 2.

BACKGROUND: Porcine parvovirus 1 (PPV1) is widespread in commercial pig farms worldwide and has a significant impact to the swine industry. Long-lasting immunity achieved by means of vaccination is the main tool to prevent PPV1 infection and its associated clinical signs. Here we evaluated the duration of immunity (DOI) conferred by a novel subunit vaccine based on the viral protein (VP) 2 of PPV1, named ReproCyc® ParvoFLEX. The DOI was assessed at 6 months post-vaccination following the standard vaccination scheme (phase I) or after re-vaccination (phase II) with a single injection administered 24 weeks after the basic vaccination scheme. A total of 46, five to six-month-old gilts, free of PPV1 and porcine reproductive and respiratory syndrome virus (PRRSV), were randomly assigned to 6 groups (three in each phase): the negative control groups were inoculated with sodium chloride (NaCl), the vaccinated groups were immunized with the PPV1 subunit vaccine and the strict controls were neither treated nor challenged. Subsequently, the negative control and vaccinated groups from each phase were challenged with a heterologous PPV1 strain. Infection of fetuses was the primary outcome parameter for efficacy, though other supportive parameters were PPV1 viremia and serological status of the gilts and the condition of their fetuses (i.e. normal, autolytic, or mummified). RESULTS: All gilts vaccinated against PPV1 tested seropositive at challenge and viremia after challenge was detectable only in the non-vaccinated animals. In this regard, fetuses positive to PPV1 by PCR were only found in litters from non-vaccinated sows. CONCLUSIONS: These results point out that the immunity developed by the PPV1 subunit vaccine is effective in terms of preventing viremia, transplacental infection of fetuses and fetal death caused by PPV1 infection. ReproCyc® ParvoFLEX was demonstrated to protect fetuses against heterologous PPV1 challenge with a DOI of 6 months after vaccination.

A novel protein chip for simultaneous detection of antibodies against four epidemic swine viruses in China.

BACKGROUND: At present, pig industry in China is faced with the complex situation of mixed infection caused by multiple pathogens. It is urgent to develop some new high-throughput molecular diagnosis assays to simultaneously detect pathogens or antibodies. Biochip array technology has made it possible to screen thousands of samples simultaneously; it has been twice named as one of the top 10 scientific and technological breakthroughs. Studies have reported encouraging results using protein biochips for detecting antibodies against avian infectious bronchitis virus and ruminant bluetongue virus, but the research of this technology for the diagnosis of swine diseases is still sparse. RESULTS: In this study, a novel protein chip was developed that can simultaneously detect the antibodies of four important swine viruses as follow, classical swine fever virus (CSFV), porcine parvovirus (PPV), Japanese encephalitis virus (JEV), and porcine reproductive and respiratory syndrome virus (PRRSV). Four prokaryotic expression plasmids pET-32a-E2 of CSFV, -VP2 of PPV, -EDIII of JEV, and -N of PRRSV were induced by IPTG (Isopropyl ß-D-1-Thiogalactopyranoside) and overexpressed in E.coli, respectively. The purified proteins were identified by Western blotting and then printed on epoxy-coated glass slides. The optimized parameters of this diagnostic chip showed that the spotting concentrations of E2、VP2、EDIII、N proteins were 0.2, 0.4, 0.4, and 0.4 mg/mL. The optimal primary and secondary antibody dilutions were 1:50 and 1: 600. Compared with the commercial ELISA (Enzyme-linked immunosorbent assay) kits, the positive and negative coincidence rates of this chip were 95.8% ~ 100 and 86.2% ~ 100%, as well as, no cross-reaction. CONCLUSION: This protein chip provided a fast, specific, and sensitive method for simultaneous detection of antibodies in clinical serum samples. Compared with traditional methods, this protein chip can monitor very small amount of serum.

Large-scale manufacture of VP2 VLP vaccine against porcine parvovirus in Escherichia coli with high-density fermentation.

Porcine parvovirus (PPV) virus-like particles (VLPs) are a potential vaccine candidate for the prevention of parvovirus-induced reproductive failure in pregnant sows. Currently, the Escherichia coli (E. coli) expression system is the most cost-efficient to express recombinant proteins. To overcome the limitations of protein misfolding and to prepare soluble highly bioactive antigen and high yields of protein, we optimized the PPV-VP2 gene, subcloned it into pET24a, pET26b, pET28a, and pET30a, and transformed it into E. coli BL21(DE3)-Tf16 competent cells. The pET28a plasmid was selected for further manipulations because it expressed high levels of the bioactive PPV-VP2 protein. Under optimal high-density fermenting conditions in a 70-L fermenter, the total yield of wet weight E. coli cells was 124.86 g/L, and PPV-VP2 protein was 2.5 g/L. After large-scale purification with Triton X-114 two-phase extraction as well as activated carbon powder adsorption, hemagglutination (HA) titers in the purified PPV-VP2 protein reached 219 and endotoxin was reduced to 2500 EU/mL. Dynamic light scattering (DLS) and transmission electron microscopy (TEM) results indicated that the purified PPV-VP2 protein self-assembled into VLPs. Immunogenicity assays in guinea pigs and pigs indicated that the ISA-201 VG adjuvanted PPV-VP2 VLP vaccine elicited hemagglutination inhibition (HI) and virus neutralization (VN) antibody titers comparable with PPV commercial inactivated vaccines, whereas viral loads in the spleen and liver of challenged guinea pigs were significantly reduced. In conclusion, our study provides a method for producing the PPV-VLP vaccine against PPV infection in E. coli and may offer a novel strategy for the soluble expression of other vaccine antigens.

An octavalent vaccine provides pregnant gilts protection against a highly virulent porcine parvovirus strain.

BACKGROUND: Porcilis® Ery+Parvo+Lepto is an octavalent inactivated ready-to-use vaccine that contains Erysipelothrix rhusiopathiae (Ery), porcine parvovirus (PPV), and six serogroups of Leptospira (Lepto). The efficacy of Porcilis® Ery + Parvo+Lepto against reproductive problems associated with porcine parvovirus (PPV) infection was evaluated in pregnant gilts. For this, a group of ninegilts was vaccinated twice (at 5 and 6 months old) with Porcilis® Ery + Parvo+Lepto (Group 1), while a group of eight gilts was included as unvaccinated controls (Group 2). All pigs were artificially inseminated 4 weeks after the second vaccination. They were challenged during early gestation with PPV-27a, a virulent cluster D strain, and euthanized to collect their offspring by hysterectomy around day 90 in pregnancy. Antibody responses against PPV in gilts were measured, and the presence of PPV in progeny was also determined. RESULTS: No clinical signs were observed after vaccination. After PPV challenge, all foetuses from the vaccinated gilts were alive (132/132), while in the unvaccinated group only 41% were alive (46/112), 19.6% were dead and 39.4% of the offspring (44/112) were mummified. PPV could be detected by qPCR in 14% of the progeny from vaccinated gilts at an average of 4.7 log10/ml, whereas this was significantly higher in the control group, where 90% of the progeny were PPV positive, with titres of 9.8 log10/ml on average. CONCLUSIONS: The present study demonstrates that vaccination of gilts with Porcilis® Ery + Parvo+Lepto was safe and induced an immune response sufficient to protect progeny against PPV by reducing transplacental infection.

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.

Porcine parvovirus VP1/VP2 on a time series epitope mapping: exploring the effects of high hydrostatic pressure on the immune recognition of antigens.

Porcine parvovirus (PPV) is a DNA virus that causes reproductive failure in gilts and sows, resulting in embryonic and fetal losses worldwide. Epitope mapping of PPV is important for developing new vaccines. In this study, we used spot synthesis analysis for epitope mapping of the capsid proteins of PPV (NADL-2 strain) and correlated the findings with predictive data from immunoinformatics. The virus was exposed to three conditions prior to inoculation in pigs: native (untreated), high hydrostatic pressure (350 MPa for 1 h) at room temperature and high hydrostatic pressure (350 MPa for 1 h) at - 18 °C, and was compared with a commercial vaccine produced using inactivated PPV. The screening of serum samples detected 44 positive spots corresponding to 20 antigenic sites. Each type of inoculated antigen elicited a distinct epitope set. In silico prediction located linear and discontinuous epitopes in B cells that coincided with several epitopes detected in spot synthesis of sera from pigs that received different preparations of inoculum. The conditions tested elicited antibodies against the VP1/VP2 antigen that differed in relation to the response time and the profile of structurally available regions that were recognized.

Comparison of immune responses induced by porcine parvovirus virus-like particles and inactivated vaccine.

Laboratory-prepared inactivated porcine parvovirus (PPV) vaccines and VP2 virus-like particles (VLPs) were utilized to immunize gilts. PPV BQ strain and SP2/0 cells were used. Hemagglutination-inhibiting (HI) antibody titers were measured in the immunized gilts and the differences in cytokine production of interferon gamma (IFN-γ, IL-2 and IL-4) were compared. CD4+ and CD8+ T cells proliferation were compared by flow cytometry. The variation between the immune response level induced by inactivated PPV vaccine and VP2 VLPs were determined. The results showed that all vaccinated gilts had HI antibody titers reaching 1:256 for at least one month post immunization and the peak level of antibody could be sustained for one month; further, PPV antibodies could be detected in the second week post immunization with VP2 VLPs. We also found that the level of cytokines (IFN-γ, IL-2 and IL-4) were all increased post immunization and continued to rise after the booster immunization; the level of increase in IFN-γ and IL-2 were significantly higher than IL-4. The flow cytometry results showed that the numbers of the CD4+ and CD8+ T cells subsets were significantly higher in the groups immunized with inactivated PPV vaccine or VP2 VLPs than those of negative control group (p<0.01); additionally, the number of CD4+ cells in the gilts that received VP2 VLP immunization was significantly higher than the inactivated vaccine group (p<0.01). In summary, the inactivated PPV vaccine and PPV VP2 VLPs were both able to induce humoral and cellular immunity, but the VP2 VLPs lead to better cellular immune responses in gilts compared to those of the inactivated vaccine..

Biology of Porcine Parvovirus (Ungulate parvovirus 1).

Porcine parvovirus (PPV) is among the most important infectious agents causing infertility in pigs. Until recently, it was thought that the virus had low genetic variance, and that prevention of its harmful effect on pig fertility could be well-controlled by vaccination. However, at the beginning of the third millennium, field observations raised concerns about the effectiveness of the available vaccines against newly emerging strains. Subsequent investigations radically changed our view on the evolution and immunology of PPV, revealing that the virus is much more diverse than it was earlier anticipated, and that some of the "new" highly virulent isolates cannot be neutralized effectively by antisera raised against "old" PPV vaccine strains. These findings revitalized PPV research that led to significant advancements in the understanding of early and late viral processes during PPV infection. Our review summarizes the recent results of PPV research and aims to give a comprehensive update on the present understanding of PPV biology.

Porcine parvovirus induces activation of NF-κB signaling pathways in PK-15 cells mediated by toll-like receptors.

Porcine parvovirus (PPV) is a pathogenic factor that primarily induces severe reproductive failure of pregnant swine, which results in extensive losses to the swine industry worldwide. In this study, a potential mechanism of PPV-induced activation of the nuclear transcription factor-kappaB (NF-κB) by infection in porcine kidney cells (PK-15) was elucidated for the first time. The subcellular localization of p65 analyzed by immunofluorescence assay (IFA) showed that PPV infection induced p65 translocation from the cytoplasm to the nucleus. p65 phosphorylation was detected in PK-15 cells with progression of PPV infection. NF-κB-regulated gene expression was enhanced in a viral dose-dependent manner using the NF-κB luciferase reporter assay system. Furthermore, PPV-induced NF-κB activation was closely related to the inhibitory kappa B alpha (IκBα) degradation. Treatment with a NF-κB-specific inhibitor demonstrated that the production of PPV progeny viruses was enhanced to some extent. In addition, these results demonstrated that the adapter molecule TIR domain-containing adapter inducing IFN-ß (TRIF) and myeloid differentiation primary-response protein 88 (MyD88)-dependent signaling pathways were involved in PPV-induced NF-κB activation. Together, these results provide evidence that the toll-like receptor (TLR) pathway participates in recognition of PPV and induction of NF-κB activation, and add to understanding of the molecular mechanisms underlying PPV infection.

[Immune Response of Recombinant Pseudorabies Virus rPRV-VP2 Expressing VP2 Gene of Porcine Parvovirus in Mice].

In order to develop a combined live vaccine that will be used to prevent against porcine parvovirus (PPV) and Pseudorabies virus (PRV) infection, the VP2 gene of PPV was inserted into the transfer vector plasmid pG to produce the recombinant plasmid pGVP2. The plasmid pGVP2 and the genome of PRV HB98 attenuated vaccine were transfected by using lipofectamine into swine testis cells for the homologous recombination. The recombinant virus rPRV-VP2 was purified by selection of green fluorescence plaques for five cycles. 6-week-old female Kunming mice were immunized intramuscularly with attenuated PRV parent HB98 strain, commercial inactivated vaccine against PPV, recombinant virus, DMEM culture solution. The injections were repeated with an equivalent dose after 2 weeks in all of the groups, and then challenged with the virulent PRV NY strain at 7 weeks after the first immunization. The recombinant virus rPRV-VP2 was successfully generated, and the recombinant virus could effectively elicite anti-PPV and PRV antibody and significant cellular immune response as indicated by anti-PPV ELISA and HI, PRV-neutralizing assay and flow cytometry. The challenge assay indicated that recombinant virus could protect the mice against the virulent PRV challenge. These results demonstrated that the recombinant virus can be a candidate recombinant vaccine strain for the prevention of PRV and PPV.

Reproduction of post-weaning multi-systemic wasting syndrome in an animal disease model as a tool for vaccine testing under controlled conditions.

Snatch farrowed, colostrum deprived piglets were inoculated with different combinations of porcine circovirus 2, porcine parvovirus and Erysipelothrix rhusiopathiae candidate vaccines. 10 piglets were mock-vaccinated. Following virus challenge with a combined porcine circovirus 2/porcine parvovirus inoculum, all animals were monitored and samples taken for serology, immunohistochemistry and qPCR. At 24 dpc all non-vaccinated animals remaining were exhibiting signs of post-weaning multi-systemic wasting syndrome which was confirmed by laboratory analysis. Details of the study, analysis of samples and performance of the candidate vaccines are described.

An inactivated whole-virus porcine parvovirus vaccine protects pigs against disease but does not prevent virus shedding even after homologous virus challenge.

Inactivated whole-virus vaccines against porcine parvovirus (PPV) can prevent disease but not infection and virus shedding after heterologous virus challenge. Here, we showed that the same is true for a homologous challenge. Pregnant sows were vaccinated with an experimental inactivated vaccine based on PPV strain 27a. They were challenged on day 40 of gestation with the virulent porcine parvovirus PPV-27a from which the vaccine was prepared (homologous challenge). On day 90 of gestation, the fetuses from vaccinated sows were protected against disease, while the fetuses of the non-vaccinated sows (control group) exhibited signs of parvovirus disease. All gilts, whether vaccinated or not vaccinated, showed a boost of PPV-specific antibodies indicative of virus infection and replication. Low DNA copy numbers, but not infectious virus, could be demonstrated in nasal or rectal swabs of immunized sows, but high copy numbers of challenge virus DNA as well as infectious virus could both be demonstrated in non-vaccinated sows.

Detection of Porcine Parvovirus 2 (Ungulate Tetraparvovirus 3) Specific Antibodies and Examination of the Serological Profile of an Infected Swine Herd.

Porcine parvovirus 2 (PPV2) is a member of a recently discovered group of swine parvoviruses occurring worldwide. It is frequently detected in lung samples suggesting some pathological role of the virus in diseases. To study this possibility an indirect ELISA was developed to detect PPV2 specific antibodies and to examine the serological profile of an infected swine herd where 185 serum samples collected from different age groups including sows were analyzed. According to the results maternal antibody levels decreased until 14 days of age and PPV2 specific antibodies started to rise between 28 to 43 days of age when respiratory signs were also observed in the examined swine herd. At 57 days of age the clinical signs disappeared and a rapid increase of PPV2 specific antibody levels could be measured simultaneously, peaking at 57 days of age. The viraemic status of different age groups was determined by qPCR using serum samples. At least a low level of viraemia was measured in every age group, but higher copy number of PPV2 was only detected at 57 days of age and the level decreased in older age groups. The changes in virus load and antibody levels together with the onset and decrease of clinical signs suggested that PPV2 had a role in the development of respiratory signs.

The adjuvanticity of ophiopogon polysaccharide liposome against an inactivated porcine parvovirus vaccine in mice.

In this study, the adjuvant activity of ophiopogon polysaccharide liposome (OPL) was investigated. The effects of OPL on the splenic lymphocyte proliferation of mice were measured in vitro. The results showed that OPL could significantly promote lymphocyte proliferation singly or synergistically with PHA and LPS and that the effect was better than ophiopogon polysaccharide (OP) at most of concentrations. The adjuvant activities of OPL, OP and mineral oil were compared in BALB/c mice inoculated with inactivated PPV in vivo. The results showed that OPL could significantly enhance lymphocyte proliferation, increase the proportion of CD4(+) and CD8(+) T cells, improve the HI antibody titre and specific IgG response, and promote the production of cytokines, and the efficacy of OPL was significantly better than that of OP. In addition, OPL significantly improved the cellular immune response compared with oil adjuvant. These results suggested that OPL possess superior adjuvanticity and that a medium dose had the best efficacy. Therefore, OPL can be used as an effective immune adjuvant for an inactivated PPV vaccine.