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A longitudinal study of cell-mediated immunity in pigs infected with porcine parvovirus.

Porcine parvovirus (PPV) is an ubiquitous pathogen causing reproductive failure in swine. Protection against reproductive failure caused by acute PPV infection has commonly been related to the presence of specific antibodies in the dam. However, the role of cell-mediated immunity during chronic PPV infection remains to be elucidated, and may be relevant to the pathogenesis of novel diseases such as postweaning multisystemic wasting syndrome (PMWS), which may be triggered by coinfection with PPV and porcine circovirus type 2 (PCV2). To investigate whether pigs infected with PPV generate a cell-mediated immune response, a longitudinal infection experiment was performed, using swine leukocyte antigens (SLA) class I characterized growing pigs (haplotype H7/H7). Pigs were intranasally inoculated with PPV at 0, 80, and 136 days. At predetermined time points, peripheral blood mononuclear cells (PBMC) were isolated, and virus-specific lymphoproliferative responses and the cytolytic activities of cytotoxic T-lymphocytes (CTL) and natural killer (NK) cells were examined. Cytolytic assays were performed by the chromium release method, using as targets a syngeneic porcine kidney cell line established for the purpose (CTL assays) and K562 cells (NK assays). A specific proliferative response of PBMC from virus-infected pigs to PPV was observed from day 101 onwards. In contrast, PBMC from mock-infected pigs did not proliferate in response to PPV. Flow cytometric analysis indicated that the CD4+CD8+ T-cell subset of PBMC proliferated in response to virus antigen, in keeping with the assumed role for these cells in immunological memory. This is, to our knowledge, the first indication of a cellular immune response following PPV infection. A weak CTL activity, which peaked on days 80 and 87, was observed in PPV-infected pigs. In vitro restimulation of PBMC with live PPV did not induce further CTL activity. A pronounced NK cell activity was detected in both virus-infected and control pigs throughout the experiment, and may have negatively affected the sensitivity of the CTL assay. In conclusion, the findings of a late lymphoproliferative response together with weak CTL activity are in keeping with an effective control of acute PPV infection by humoral immunity, but open the possibility that cellular immunity may play a role in controlling PPV reinfection. Finally, we find that the established experimental model using SLA characterized pigs may constitute a valuable tool for future studies of CTL activity in pigs.

Animals↗

The NS and capsid genes determine the host range of porcine parvovirus.

Porcine parvovirus is an autonomous parvovirus which normally infects pigs and multiplies in porcine cells in vitro. In this report, we describe the properties of a variant designated P2, which has extended its host range to include canine cells. The variant was able to produce cytopathic effects (CPE) in canine cells, unlike the prototype NADL-2 strain. The variant also produced higher viral antigen and infectivity titers in canine cells than the NADL-2 strain, whereas both strains produced CPE and similar titers in porcine cells. Generation of recombinant plasmids between the P2 variant DNA and an infectious clone of NADL-2, and analysis of the properties of the virus stocks produced from these recombinant plasmids, indicated that two changes were necessary for this extension in the host range. One change was located in the nonstructural protein coding region and the other in the capsid coding region.

Amino Acid Sequence↗

Interepizootic survival of porcine parvovirus.

Porcine parvovirus (PPV) was transmitted by direct contact between experimentally infected and susceptible pigs at 1 and 2 weeks, but not at 4, 8, 16, or 25 weeks, after experimental infection. In contrast, PPV was found to remain infectious for at least 14 weeks in uncleaned rooms previously vacated by experimentally infected pigs. These findings suggest that facilities contaminated by secretions and excretions of infected pigs may provide the major means by which PPV survives between episodes of acute infection.

Animals↗

Reproductive disease experimentally induced by exposing pregnant gilts to porcine parvovirus.

Porcine parvovirus (PPV) was administered intravenously or intranasally and orally between the 22nd and the 81st days of gestation to 20 pregnant gilts that were free of hemagglutination-inhibiting (HI) antibody for PPV. Gilts were exposed to 1 or both of 2 strains (NADL-7, NADL-2) of PPV and were killed 21 or more days later. Fetal and maternal fluids and tissues collected at necropsy were tested for PPV, viral antigen, and HI antibody. Transplacental infection occurred with 11 of 12 gilts given the mixture of strains NADL-7 and NADL-2 and with the 1 gilt given strain NADL-7 alone, but not with any of the 7 gilts given strain NADL-2 alone. The 8 noninfected litters were comprised of 74 fetuses of which 73 were alive and 1 was dead. The 12 infected litters were comprised of 91 fetuses of which 62 were alive (31 infected) and 29 were dead (26 infected). Virus was isolated from all of the 57 infected fetuses. Viral antigen was detected in tissues of 50 fetuses, including 5 live fetuses and 24 dead fetuses that were laden with antigen. All dead fetuses with a high concentration of viral antigen in their tissues were members of litters of gilts that were exposed to PPV no later than the 42nd day of gestation. Antibody was detected in serums (HI titers 10 through 1,280) of 27 of the 31 live infected fetuses and in serum (HI titer 10) of 1 fetus for which infection was not demonstrated. All serums collected from gilts at necropsy contained antibody (HI titers 320 through 2560) for PPV, but with the exception of isolating virus from 1 uterine lymph node, neither virus nor antigen was detected in maternal tissues.

Animals↗

The effect of porcine parvovirus and porcine reproductive and respiratory syndrome virus on porcine reproductive performance.

From a worldwide perspective, porcine parvovirus (PPV) and porcine reproductive and respiratory syndrome virus (PRRSV) are the most common viral causes of porcine reproductive failure. A typical epidemic of PPV-induced reproductive failure is presented as an increased number of mummified fetuses and sometimes, entire litters are mummified. If infection with PPV is very early in gestation, the number of liveborn pigs may be further reduced as a result of embryonic death and resorption. During the acute stage of infection gilts and sows have few, if any, clinical signs, and it is unlikely that PPV is ever the direct cause of abortion. In contrast, a typical epidemic of PRRSV-induced reproductive failure is presented as a broader spectrum of clinical features including abortions, late-term dead fetuses, stillborn pigs, and weakborn pigs. In the later stages of an epidemic, there may also be an increase in the number of mummified fetuses, but their prevalence is likely to be far less than during an epidemic of PPV-induced reproductive failure. During the acute stage of infection with PRRSV, gilts and sows may have few, if any, clinical signs, or they may be severely affected and even die. This difference largely reflects the relative virulence of the strain of PRRSV causing the epidemic. A timely and reliable laboratory diagnosis of either disease can be made when appropriate tests are performed with appropriate samples. Vaccines are available for prevention of both diseases.

Animals↗

Multiplex PCR for rapid detection of pseudorabies virus, porcine parvovirus and porcine circoviruses.

A multiplex PCR (mPCR) assay was developed and subsequently evaluated for its effectiveness as a means to simultaneously detect multiple viral infections of swine. Specific primers for each of four common DNA viruses, namely, pseudorabies virus (PRV), porcine circovirus type I (PCV1), porcine circovirus type II (PCV2), and porcine parvovirus (PPV), were used for testing procedure. The assay was shown to be highly sensitive in that as little as 10(-4) ng of each of the respective amplicons (approximately equal to 10,000 molecules) was detected when a composite of all four viruses (including both field and gene-deleted permutations of PRV) was tested as a single sample. It was also effective for detecting one or more of these same viruses in various combinations in specimens including lymph nodes, lungs, spleens, and tonsils collected from clinically ill pigs, and in specimens in spleen collected from aborted fetuses. The relative efficiency (compared to performing separate assays for each virus) and apparent sensitivity of mPCR suggest its potential application for routine molecular diagnostic purposes.

Animals↗

Detection of porcine circovirus type 2, porcine parvovirus and porcine pseudorabies virus from pigs with postweaning multisystemic wasting syndrome by multiplex PCR.

Multiplex PCR was established to detect porcine circovirus type 2 (PCV-2), porcine parvovirus (PPV) and porcine pseudorabies virus (PRV) and applied to samples from 137 piglets exhibiting clinical signs of postweaning multisystemic wasting syndrome (PMWS). PCV-2 DNA was detected from all samples. Moreover, 43 samples were positive for PPV but negative for PRV; 11 samples were positive for PRV but negative for PPV; and 35 samples were positive both for PPV and PRV. These results suggests that PCV-2 co-infection with PRV and PPV may play an important role in PMWS. Also, multiplex PCR is an appropriate candidate method for diagnosis of PCV-2, PRV and PPV simultaneously in field cases.

Animals↗

Pseudorabies virus, porcine parvovirus, and porcine enterovirus interactions with the zona pellucida of the porcine embryo.

Porcine embryos (n = 93) were incubated on cell monolayers that had been previously inoculated with pseudorabies virus, porcine parvovirus (PPV), or each of 2 porcine enteroviruses. After 2, 24, or 48 hours of incubation, the embryos were fixed in glutaraldehyde and examined by electron microscopic procedures. It was found that pseudorabies virus adsorbed to the zona pellucida (ZP) and entered sperm tracks in the ZP. The PPV and both enteroviruses entered pores in the ZP and were associated with sperm that were at or near the outer surface of the ZP. In addition, PPV was seen enmeshed in cellular debris on the outer surface of the ZP. Evidence of a productive viral infection of the blastomeres of the embryos was not found.

Animals↗

Distribution of porcine parvovirus in porcine circovirus 2-infected pigs with postweaning multisystemic wasting syndrome as shown by in-situ hybridization.

In-situ hybridization with a nonradioactive digoxigenin-labelled probe was used to study the distribution of porcine parvovirus (PPV) in formalin-fixed paraffin wax-embedded tissues from 10 porcine circovirus 2 (PCV2)-infected weaned pigs with naturally occurring postweaning multisystemic wasting syndrome (PMWS). A 226 base pair DNA fragment from a VP2 structural gene was generated by polymerase chain reaction (PCR) and used as a probe. Lymph node, spleen, thymus and tonsil were positive by PCR, demonstrating the presence of PPV DNA in the tissue samples from four of 10 pigs tested. PPV nucleic acid was also detected consistently in lymph node, spleen, thymus and tonsil by in-situ hybridization. Detection of PPV DNA from PCV2-infected pigs with PMWS suggests that PPV also plays a role in the pathogenesis of PMWS.

Animals↗

[Prevalence and association of porcine circovirus type 2 (PCV2), porcine parvovirus (PPV) and porcine reproductive and respiratory syndrome virus (PRRSV) in aborted fetuses, mummified fetuses, stillborn and nonviable neonatal piglets].

Porcine circovirus type 2 (PCV2) seems to cause reproductive failure in sows not only in experimental studies. A retrospective study was made with a total of 252 aborted fetuses, mummified fetuses, stillborn and nonviable neonatal piglets to determine the presence of PCV2, porcine parvovirus (PPV) and porcine respiratory and reproductive syndrome virus (PRRSV) by PCR. PCV2 was found in all stages of gestation in 27.1 percent of samples examined. A statistically significant association could be shown between the detection of PCV2 and PRRSV. However, no significant association was seen between the detection of PCV2 and PPV and between PPV and PRRSV.

Abortion, Veterinary↗

KBSH parvovirus: comparison with porcine parvovirus.

We compared the molecular, antigenic, and pathogenic properties of KBSH parvovirus to those of porcine parvovirus (PPV) isolate NADL-8. KBSH, propagated in swine testes cells in culture, possessed two major capsid polypeptides of 83 and 64 kilodaltons that were similar in size to those of PPV. KBSH-infected cells also contained an 86-kilodalton nonstructural polypeptide that was identical in size to the PPV nonstructural polypeptide (NS-1). The KBSH polypeptides were structurally similar but not identical to the corresponding PPV polypeptides, as revealed by partial proteolysis mapping. Viral replicative-form DNA from KBSH-infected cells was similar in size to PPV replicative-form DNA and exhibited similar but not identical restriction endonuclease cleavage patterns to that of PPV replicative-form DNA. Antigenically, the two viruses were also very closely related. By using heterologous and homologous antisera, the two viruses were indistinguishable in hemagglutination inhibition and immunoprecipitation assays. However, pathogenically these viruses were dramatically different. NADL-8 caused fetal death when injected into swine fetuses in utero and viremia and high persisting antibody titers when administered orally to weaning-age swine. KBSH-inoculated fetuses were normal in appearance, and pigs orally exposed to KBSH failed to establish viremia and demonstrated only transient antibody titers. Thus, KBSH appears to be a PPV that is very closely related to a highly pathogenic PPV isolate, yet is itself nonpathogenic in swine. This reduced pathogenic potential of KBSH may be attributable to its poor ability to replicate in swine.

Animals↗

Studies on the multiplication of a porcine parvovirus.

A porcine parvovirus has been characterized with regard to its replication in foetal porcine kidney cells and certain biophysical properties. Electron microscopy of infected cells at selected times postinfection revealed that porcine parvovirus replication took place within or near a series of granular intranuclear inclusions which may be contiguous with cellular heterochromatin. Developing virions were observed to aggregate into a nucleolar-like amorphous mass which gradually disrupted as cellular integrity was lost. Purified virions were found to have a buoyant density in CsCl of 1.38 g/ml, while 'empty' particles had a buoyant density of 1.29 g/ml. The particle diameter was calculated to be approximately 22 nm.

Animals↗

Large scale production and downstream processing of a recombinant porcine parvovirus vaccine.

Porcine parvovirus (PPV) virus-like particles (VLPs) constitute a potential vaccine for prevention of parvovirus-induced reproductive failure in gilts. Here we report the development of a large scale (25 l) production process for PPV-VLPs with baculovirus-infected insect cells. A low multiplicity of infection (MOI) strategy was efficiently applied avoiding the use of an extra baculovirus expansion step. The optimal harvest time was defined at 120 h post-infection at the MOI used, with the cell concentration at infection being 1.5x10(6) cells/ml. An efficient purification scheme using centrifugation, precipitation and ultrafiltration/diafiltration as stepwise unit operations was developed. The global yield of the downstream process was 68%. Baculovirus inactivation with Triton X-100 was successfully integrated into the purification scheme without an increase in the number of process stages. Immunogenicity of the PPV-VLPs tested in guinea pigs was similar to highly purified reference material produced from cells cultured in the presence of serum-containing medium. These results indicate the feasibility of industrial scale production of PPV-VLPs in the baculovirus system, safety of the product, and the potency of the product for vaccine application.

Animals↗

Effect of porcine parvovirus vaccination on the development of PMWS in segregated early weaned pigs coinfected with type 2 porcine circovirus and porcine parvovirus.

The objectives of this study were to determine if coinfection of segregated early weaned (SEW) pigs with porcine circovirus type 2 (PCV2) and porcine parvovirus (PPV) induces an increase in the incidence of post-weaning multisystemic wasting syndrome (PMWS) compared to singular PCV2 infection, and to determine if vaccination against PPV protects pigs against PMWS associated with PCV2/PPV coinfection in SEW pigs. Seventy, 3-week-old, SEW pigs were randomly assigned to one of the five groups. Pigs in group 1 (n = 14) served as the negative controls, group 2 pigs (n = 14) were inoculated with PCV2, group 3 pigs (n = 12) were inoculated with PPV, groups 4 (n = 16) and 5 (n = 14) pigs were inoculated with both PCV2 and PPV. Pigs in groups 1-3 and 5 were vaccinated with two doses of a killed parvovirus-leptospira-erysipelothrix (PLE) vaccine prior to inoculation. The PCV2/PPV-coinfected pigs (groups 4 and 5) had significantly (P < 0.05) higher and more persistent fevers than the singular PCV2-infected pigs. One pig in each of the coinfected groups developed clinical disease (fever, respiratory disease, jaundice, weight loss) consistent with PMWS. Lymphoid depletion was significantly (P < 0.05) more severe in the dually-infected pigs at 42 days post-inoculation (DPI). Vaccinated, coinfected pigs (group 5) remained viremic significantly (P < 0.05) longer and had higher copy numbers of genomic PCV2 DNA in sera at 28, 35, and 42 DPI compared to the unvaccinated coinfected pigs (group 4). PPV-viremia was detected only in the unvaccinated group 4 pigs. PLE-vaccination prevented PPV-viremia but did not prevent clinical PMWS or reduce the severity of lymphoid depletion in PCV2/PPV-coinfected pigs. Evidence of increased incidence of clinical PMWS due to vaccination was not observed in this model.

Animals↗

In vitro exposure of preimplantation porcine embryos to porcine parvovirus.

Early porcine embryos at the four- to eight-cell stage can be infected with either the virulent (NADL-8) or avirulent KBSH strain of porcine parvovirus (PPV) by microinjection or by incubation of embryos with virus. Treatment of embryos by microinjection of virus or incubation in media with virus did not significantly inhibit in vitro development of the embryos when compared with untreated controls. RNA-DNA hybridization was used to identify the presence of virus associated with embryos. It was found that PPV-DNA was present in viable embryos after microinjection of embryos with KBSH and NADL-8 strains of PPV and after incubation of embryos with KBSH strain. The data indicated the presence of replicative virus associated with viable porcine embryos.

Journal Article↗

Porcine parvovirus: natural and experimental infections of the porcine fetus and prevalence in mature swine.

Antibodies against porcine parvovirus were detected in 17 of 116 prenursing pig sera. Antibodies against transmissible gastroenteritis or ECPO-6 (an enterovirus) were not detected in prenursing sera of the pigs tested. Seventy-seven percent of 129 serum samples from 23 Ohio farms and 82% of 96 samples from slaughter plants in Ohio were serologically positive for porcine parvovirus. Mummies or other abnormalities were not observed in newly born pigs exposed to porcine parvovirus by the transuterine route 101 days after gestation. Indirect evidence suggested that the virus had not spread to other fet uses following exposure after 101 days at least not in a sufficient amount of time to stimulate detectable antibody. Direct intrafetal exposure to porcine parvovirus (i.m. injection, transutero) after 62 days of gestation resulted in dealth and mummification of the two fetuses, and apparently in the subsequent spread of the virus, as five of nine live pigs born were serologically positive for porcine parvovirus and these five pigs had not been injected with the virus. Immunoglobulin G was detected in all newborn pigs irregardless of known antigenic stimulation or the presence of specific antibody. In general, the presence of immunoglobulin M or immunoglobulin A in fetal serum was correlated with a history of antigenic stimulation or the presence of detectable antibody.

Animals↗

Development of hybridization probes on the basis of recombinant DNA to identify porcine parvoviruses and rotaviruses.

Porcine parvovirus (PPV) and porcine rotavirus (PRV) infections can be diagnosed, and the diagnosis greatly contributes to their control. For the diagnosis of PPV and PRV of pigs recombinant DNA were obtained which could be used as hybridization probes. Homology between PPV-RNA and cloned cDNA was confirmed, and the ability of plasmid probes to detect PRV was demonstrated. Hybridization with preparations of both PPV-infected cells and purified PPV gave positive reproducible results. The results are useful as an experience for the development of a sandwich hybridization system for PPV detection.

Animals↗

A slide latex agglutination test for the rapid detection of antibodies in serum against porcine parvovirus.

A slide latex agglutination test (LAT) was developed and evaluated to detect serum antibodies against porcine parvovirus. Porcine parvovirus antigen was obtained by 10% PEG-6000 and 0.5 mol/l sodium chloride precipitation, and inactivated by 0.1% methanal. Two per cent suspensions of latex particles (0.5-0.8 microm) were coated by adding an equal volume of porcine parvovirus antigen at 0.34 microg/ml. Repeatability of latex agglutination test was evaluated with a panel of 100 sera using the same and different antigen lots. A good agreement between LAT and haemagglutination inhibit assay was observed. Because of convenience and speed of performance, this method would be used widely in clinic examination.

Animals↗