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A novel recombinant virus-like particle vaccine for prevention of porcine parvovirus-induced reproductive failure.

A novel vaccine against porcine parvovirus (PPV), composed of recombinant virus-like particles (PPV-VLPs) produced with the baculovirus expression vector system (BEVS) at industrial scale, was tested for its immunogenicity and protective potency. A formulation of submicrogram amounts of PPV-VLPs in a water-in-mineral oil adjuvant evoked high serum antibody titres in both guinea pigs, used as reference model, and target species, pigs. A single immunisation with 0.7microg of this antigen yielded complete foetal protection against PPV infection after challenge with a virulent strain of this virus. Furthermore, also in the presence of mild adjuvants the protective action of these PPV-VLPs is excellent. This recombinant subunit vaccine overcomes some of the drawbacks of classical PPV vaccines.

Adjuvants, Immunologic↗

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↗

A real-time PCR assay for rapid detection and quantitation of canine parvovirus type 2 in the feces of dogs.

We describe a rapid, sensitive and reproducible real-time PCR assay for detecting and quantifying canine parvovirus type 2 (CPV-2) DNA in the feces of dogs with diarrhea. An exogenous internal control was added to control the assay performance from extraction to amplification. The method was demonstrated to be highly specific and sensitive, allowing a precise CPV-2 DNA quantitation over a range of eight orders of magnitude (from 10(2) to 10(9) copies of standard DNA). The reproducibility of the CPV-2 real-time PCR assay was assessed by calculating the coefficients of variation (CV) intra-assay and inter-assay for samples containing amounts of CPV-2 DNA spanning the whole range of the real-time PCR standard curve. Then, fecal specimens from diarrheic dogs were analyzed by hemagglutination (HA), conventional PCR and real-time amplification. Comparison between these different techniques revealed that real-time PCR is more sensitive than HA and conventional gel-based PCR, allowing to detect low viral titers of CPV-2 in infected dogs.

Animals↗

Experimental infection of 3-week-old conventional colostrum-fed pigs with porcine circovirus type 2 and porcine parvovirus.

This report describes an experimental infection with porcine circovirus type 2 (PCV2) in combination with porcine parvovirus (PPV) in 3-week-old conventional colostrum-fed pigs with maternal antibodies to both viruses. Two groups of four pigs each were inoculated with PCV2 and PPV. One of the groups received also a commercial inactivated vaccine against porcine pleuropneumonia to evaluate possible effects of the stimulation of the immune system of pigs on the infection. Another group of four pigs was kept as uninfected control. Clinical signs, rectal temperatures and body weights were recorded. Serum antibody titers to PCV2 and PPV were determined at weekly intervals. Pigs were killed 42 days after inoculation and tissue samples were examined for the presence of gross and microscopic lesions. Tissues were also analyzed for the presence of PCV2 and PPV DNA by PCR, and for the presence of PCV2 antigen by immunohistochemistry (IHC). All the pigs had serum antibodies to PCV2 and PPV at the beginning of the trial. None of them developed clinical symptoms or pathological lesions typical of post-weaning multisystemic wasting syndrome (PMWS), a disease associated to PCV2 infection. However, IHC and/or PCR analyses showed that clinically silent PCV2 infection developed in five of the eight inoculated pigs, regardless of the administration of the vaccine. In particular, PCV2 DNA and/or antigen were detected in most of the tissues examined in the two pigs with the lowest titer of maternal PCV2 antibodies at the beginning of the trial. PPV DNA was not detected in any of the samples examined. The five pigs with PCR and/or IHC evidence of PCV2 infection had a mean weight gain during the experiment lower than that of the inoculated PCR-negative pigs considered together and that of the control pigs. In conclusion, it would appear that passive immunity against PCV2 can play a role in preventing the development of PMWS, but is not able to prevent the establishing of clinically silent PCV2 infections. The dissemination and persistence of the virus in the tissues may depend on the level of PCV2 antibodies at the time of inoculation.

Adjuvants, Immunologic↗

Evolution of canine parvovirus--a need for new vaccines?

Canine parvovirus (CPV) is a new virus, which is continuing to evolve, giving rise to new antigenic types and virus mutants that spread through the dog population. The most successful mutants, from an evolutionary perspective, appear to be selected by improved binding to the CPV receptor, the canine transferrin receptor, and by an extended host range, which for the newer antigenic types now includes both the dog and the cat. The new viruses also show antigenic differences that can be defined by binding of certain monoclonal antibodies; they also differ in their reactivity in virus neutralisation tests, using immune sera raised against the various antigenic types. These differences may influence the susceptibility of young animals to infection at the time when the level of maternally derived antibody decreases to the minimum protective titre. This minimum protective titre may vary depending on the infecting virus type. There is, however, a high degree of cross-protection between the virus types and the true relevance of the differences in neutralisation titer is currently not known.

Animals↗

Tissue distribution of the antigenic variants of canine parvovirus type 2 in dogs.

Twelve dogs dead as consequence of natural infection caused by canine parvovirus (CPV) type 2a (n=4), type 2b (n=4) or type 2c (n=4) were investigated for determining the viral DNA loads in different tissue samples. By means of a real-time PCR assay, CPV DNA was detected in all tissues examined, with the highest titres observed in the lymphoid tissue and the lowest loads in the urinary tract. Surprisingly, the nervous tissue was found to contain considerable amounts of CPV nucleic acid. Similar patterns of tissue distribution were observed in all the examined dogs irrespective of the antigenic variant causing the disease.

Animals↗

Release of canine parvovirus from endocytic vesicles.

Canine parvovirus (CPV) is a small nonenveloped virus with a single-stranded DNA genome. CPV enters cells by clathrin-mediated endocytosis and requires an acidic endosomal step for productive infection. Virion contains a potential nuclear localization signal as well as a phospholipase A(2) like domain in N-terminus of VP1. In this study we characterized the role of PLA(2) activity on CPV entry process. PLA(2) activity of CPV capsids was triggered in vitro by heat or acidic pH. PLA(2) inhibitors inhibited the viral proliferation suggesting that PLA(2) activity is needed for productive infection. The N-terminus of VP1 was exposed during the entry, suggesting that PLA(2) activity might have a role during endocytic entry. The presence of drugs modifying endocytosis (amiloride, bafilomycin A(1), brefeldin A, and monensin) caused viral proteins to remain in endosomal/lysosomal vesicles, even though the drugs were not able to inhibit the exposure of VP1 N-terminal end. These results indicate that the exposure of N-terminus of VP1 alone is not sufficient to allow CPV to proliferate. Some other pH-dependent changes are needed for productive infection. In addition to blocking endocytic entry, amiloride was able to block some postendocytic steps. The ability of CPV to permeabilize endosomal membranes was demonstrated by feeding cells with differently sized rhodamine-conjugated dextrans together with the CPV in the presence or in the absence of amiloride, bafilomycin A(1), brefeldin A, or monensin. Dextran with a molecular weight of 3000 was released from vesicles after 8 h of infection, while dextran with a molecular weight of 10,000 was mainly retained in vesicles. The results suggest that CPV infection does not cause disruption of endosomal vesicles. However, the permeability of endosomal membranes apparently changes during CPV infection, probably due to the PLA(2) activity of the virus. These results suggest that parvoviral PLA(2) activity is essential for productive infection and presumably utilized in membrane penetration process of the virus, but CPV also needs other pH-dependent changes or factors to be released to the cytoplasm from endocytic vesicles.

Amiloride↗

Human Parvovirus B19 nonstructural protein transactivates the p21/WAF1 through Sp1.

The expression of human Parvovirus B19 nonstructural protein 1 (NS1) induces cell cycle arrest at the G1 phase and is accompanied by increased expression of the cyclin-dependent kinase inhibitor, p21/WAF1. Here, we provide direct evidence that NS1 mediates the transactivation of p21/WAF1. Up-regulation of p21/WAF1 by wild-type NS1 but not an NS1 mutant deleted of its NTP binding motif was observed. We also demonstrated that the wild-type NS1 is unable to induce G1 arrest in p21-deficient cells. Using reporter plasmids containing various mutants of the p21/WAF1 promoter, luciferase assay further revealed that the binding sites of the promoter to the transcription factor Sp1 are critical for NS1-mediated transactivation. Indeed Sp1 interacts only with the wild-type NS1 but not the NS1 mutant. These results indicate a cooperative contribution of NS1 and Sp1 to the transactivation of p21/WAF1, which leads to G1 arrest.

Base Sequence↗

Induction of neutralizing antibodies by a tobacco chloroplast-derived vaccine based on a B cell epitope from canine parvovirus.

The 2L21 epitope of the VP2 protein from the canine parvovirus (CPV), fused to the cholera toxin B subunit (CTB-2L21), was expressed in transgenic tobacco chloroplasts. Mice and rabbits that received protein-enriched leaf extracts by parenteral route produced high titers of anti-2L21 antibodies able to recognize the VP2 protein. Rabbit sera were able to neutralize CPV in an in vitro infection assay with an efficacy similar to the anti-2L21 neutralizing monoclonal antibody 3C9. Anti-2L21 IgG and seric IgA antibodies were elicited when mice were gavaged with a suspension of pulverized tissues from CTB-2L21 transformed plants. Combined immunization (a single parenteral injection followed by oral boosters) shows that oral boosters help to maintain the anti-2L21 IgG response induced after a single injection, whereas parenteral administration of the antigen primes the subsequent oral boosters by promoting the induction of anti-2L21 seric IgA antibodies. Despite the induced humoral response, antibodies elicited by oral delivery did not show neutralizing capacity in the in vitro assay. The high yield of the fusion protein permits the preparation of a high number of vaccine doses from a single plant and makes feasible the oral vaccination using a small amount of crude plant material. However, a big effort has still to be done to enhance the protective efficacy of subunit vaccines by the oral route.

Administration, Oral↗

Human parvovirus B19 infection of monocytic cell line U937 and antibody-dependent enhancement.

Human parvovirus B19 (B19) infects human erythroid lineage cells. Accumulating evidence also shows that B19 is detectable in nonerythroid lineage cells in vivo, but the mechanism of infection is still not clear. In this study, we explored the mode of B19 infection of human monocytic cell line U937. An in vitro infection study demonstrated B19 binding of U937 and slow replication of B19-DNA with B19-NS1 mRNA transcription. B19-DNA replication in U937 was accompanied by undetectable level of B19-VP1 mRNA transcription, indicating that B19 infection of U937 cells may be abortive. Levels of B19-DNA and B19-NS1 mRNA transcription increased in the presence of anti-B19 IgG antibodies, but this effect decreased in the presence of anti-Fc receptor antibodies, showing antibody-dependent enhancement by B19 infection. Antibody-dependent enhancement also caused the increased production of TNFalpha in U937. This study is the first to suggest B19 infection of nonerythroid lineage cells with antibody-dependent enhancement.

Antibodies, Viral↗

Parvovirus B19 genome as a single, two-state replicative and transcriptional unit.

The variation in the amount of parvovirus B19 DNA and different classes of RNA in permissive and non-permissive infected cells was analysed by means of quantitative real-time PCR and RT-PCR assays. In the permissive bone marrow mononuclear cells, UT7/Epo and KU812Ep6 cells, viral DNA usually increased within 48 hpi, rarely exceeding 2 Logs with respect to input DNA. Viral RNA was always present within 2-6 hpi, its increase paralleled that of viral DNA up to 36-48 hpi, and all the different classes of viral RNA were constantly represented in stable relative amounts throughout the infection cycle. In the non-permissive TF-1 cells, viral DNA did not increase and only one most represented single class of viral RNA was detected. Our data do not support the current model for B19 virus replication and transcription, consisting in different early and late expression patterns, but suggest an alternative model, indicating that the B19 virus genome should be considered a single, two-state replicative and transcriptional unit.

Bone Marrow Cells↗

Survival of parvovirus B19-infected cells by cellular autophagy.

Human parvovirus B19 (B19) is a well-known pathogenic agent which causes apoptosis in erythrocyte lineage cells. Here, we provide the first evidence that mitochondrial autophagy is specifically found in the B19-infected cells. The protein expression ratio for LC3-II/LC3-I increased significantly in infected cells, indicating possible involvement of cellular autophagy in the infection process. Immunofluorescence confocal microscopy analyses revealed that B19 infection induced an intracellular autophagosome as judged by endogenous LC3 staining. Moreover, inhibition of autophagy by 3-MA significantly facilitated B19-infection-mediated cell death. These results suggest a novel mechanism by which B19-infected cells survive by cellular autophagy.

Adenine↗

Different mechanisms of antibody-mediated neutralization of parvoviruses revealed using the Fab fragments of monoclonal antibodies.

Antibody binding and neutralization are major host defenses against viruses, yet the mechanisms are often not well understood. Eight monoclonal antibodies and their Fab fragments were tested for neutralization of canine parvovirus and feline panleukopenia virus. All IgGs neutralized >85% of virus infectivity. Two Fabs neutralized when present at 5 nM, while the others gave little or no neutralization even at 20-100 nM. The antibodies bind two antigenic sites on the capsids which overlap the binding site of the host transferrin receptor (TfR). There was no specific correlation between Fab binding affinity and neutralization. All Fabs reduced capsid binding of virus to purified feline TfR in vitro, but the highly neutralizing Fabs were more efficient competitors. All partially prevented binding and uptake of capsids by feline TfR on cells. The virus appears adapted to allow some infectivity in the presence of at least low levels of antibodies.

Animals↗

Frequency of transmission of human parvovirus B19 infection by fibrin sealant used during thoracic surgery.

BACKGROUND: Fibrin sealant is used in many kinds of surgical procedures. Although pasteurization is insufficient to remove human parvovirus (HPV) B19 from this plasma-derived product, the frequency of HPV B19 infection transmitted by its use has never been known. METHODS: Blood samples of 85 patients more than 20 years of age who had undergone pulmonary resection with fibrin sealant were obtained before and 12, 24, and 48 weeks after surgery. Anti-HPV B19 antibody IgG (HPV B19 IgG) and HPV B19 DNA were detected with these samples. RESULTS: In 56 (65.9%) of 85 patients, blood samples obtained before operation were positive for HPV B19 IgG. In these 56 patients, blood samples obtained 12 to 48 weeks after surgery were all negative for HPV B19 DNA by polymerase chain reaction (PCR). In 6 (20.7%) of 29 patients whose blood samples were negative for HPV B19 IgG before surgery, blood samples obtained 12 to 48 weeks after surgery were positive for HPV B19 DNA by PCR and also positive for HPV B19 IgG. In 5 of these 6 patients reticulocyte counts decreased to less than 10 x 10(9)/l 12 to 20 days after surgery. CONCLUSIONS: Epidemiologic evidence suggests that more than 20% uninfected persons were subsequently infected with HPV B19 by use of fibrin sealant during surgery.

Adult↗

Genetic heterogeneity of the immunogenic viral capsid protein region of human parvovirus B19 isolates obtained from an outbreak in a pediatric ward.

Whereas human parvovirus B19 commonly infects children and causes erythema infectiosum, it causes more severe diseases when it infects adults. In order to examine whether different clinical outcomes of B19 infection can be ascribed to the viral genetic heterogeneity, we have determined the nucleotide sequence of highly immunogenic portions of the B19 genome obtained from six patients with various clinical manifestations in a single outbreak. Our observations demonstrated that although the B19 sequences showed a significant heterogeneity, it was not correlated with the clinical manifestation. It was thus suggested that the host immune response to B19 infection may be a major determinant of clinical presentations associated with acute B19 infection.

Adult↗

Acute splenic sequestration together with aplastic crisis caused by human parvovirus B19 in patients with sickle cell disease.

Three children with homozygous sickle cell disease, 22 months and 8 and 10 years of age, had clinical and hematologic manifestations of aplastic and splenic sequestration crisis simultaneously. They had an acute drop in hemoglobin level (16, 20, and 45 gm/L), reticulocytopenia (0.1%, 0.6%, and 0.3%), and sudden splenic enlargement. Evidence of recent parvovirus infection was demonstrated.

Anemia, Aplastic↗

Human parvovirus B19-associated arthritis in children.

Human parvovirus B19 (HPV B19) infection has been associated with chronic joint complaints in adult patients. We now report 22 children with joint complaints associated with recent HPV B19 infection. These children had either erythema infectiosum or serologic evidence of recent infection. Twenty children had arthritis; two had arthralgias. Eleven children had associated constitutional symptoms. Laboratory findings were generally normal. The duration of joint symptoms was less than 4 months in 14 children; however, six children have had persistent arthritis for 2 to 13 months, which would fulfill criteria for the diagnosis of juvenile rheumatoid arthritis. Although HPV B19 is usually associated with acute arthritis of brief duration, in some children infection with HPV B19 may be associated with the development of chronic arthritis.

Adolescent↗

Prospective evaluation of 618 pregnant women exposed to parvovirus B19: risks and symptoms.

OBJECTIVE: To assess the risk of maternal parvovirus B19 infection from exposure to various sources and the fetal morbidity of those infections. METHODS: We obtained demographic and occupational information about pregnant women exposed to sources of B19 and about the nature and duration of the exposures. We performed serologic testing 10-14 days after exposure using an indirect capture enzyme-linked immunosorbent assay. Women with immunoglobulin (Ig) M were examined with weekly ultrasound until 12 weeks after exposure, and the outcome of the pregnancy was ascertained from interviews with patients and their obstetricians. Logistic regression analysis was used to determine risk factors for maternal immunity and infection by B19. RESULTS: Of 618 pregnant women exposed, 307 (49.7%) were immune to B19, 259 remained susceptible after exposure, and 52 (16.7% of all susceptibles) contracted B19 infection. None of the 52 fetuses of infected women developed nonimmune hydrops, and there were no fetal deaths attributable to B19 in this group. The relative risk of maternal B19 infection was 2.8 if the source was a related child living in the household (95% confidence interval 1.7, 4.6; P < .001). No significant differences were found for maternal B19 infection in eight categories of maternal occupation. Maternal symptoms of polyarthralgia (46%), fever (19%), and nonspecific rash (38%) were significantly more common (P < .001) in IgM-positive patients than in noninfected women (4.1%, 2.8%, and 5.7%, respectively). Only 17 (33%) of the IgM-positive women were entirely asymptomatic. CONCLUSION: The risk of maternal B19 infection in pregnancy could not be predicted by a gravida's occupation, but it was significantly higher when the source of exposure was her own child. The fetal risk of nonimmune hydrops after maternal B19 infection must be very low. As a consequence, exclusion of pregnant women from the workplace during endemic periods with seasonal clusters of cases is not justified. Weekly fetal ultrasound evaluation in these cases carries a low yield.

Adult↗