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PubMed2003

Ginseng extract in aluminium hydroxide adjuvanted vaccines improves the antibody response of pigs to porcine parvovirus and Erysipelothrix rhusiopathiae.

Ginseng, the dry extract prepared from the Panax ginseng C.A. Mayer-root contain immunomodulators named ginsenosides, which in the pig enhance the antibody response to viral and bacterial antigens. The enhancing effect of ginseng was demonstrated vaccinating pigs against porcine parvovirus (PPV) and Erysipelothrix rhusiopathiae infections, using commercially available vaccines. The potency of the licensed, aluminium hydroxide adjuvanted; vaccines were compared with those supplemented with ginseng. The antibody response to PPV was measured by the haemagglutination inhibition (HI) test whereas the mouse potency test and ELISA evaluated the immune response to E. rhusiopathiae. Antibodies to the 64-66 kDa glycoprotein of the E. rhusiopathiae were demonstrated by immunoblotting. The qualitative antibody responses were evaluated by means of ELISA(s) using monoclonal antibodies to swine IgG1 and IgG2. The addition of 2mg ginseng per vaccine dose, potentiate the antibody response of the commercial vaccines without altering their safety. Significantly higher (P<0.001) antibody titres were achieved to both PPV and to E. rhusiopathiae by the supplementation with ginseng. Aluminium hydroxide adjuvanted vaccines favoured the production of IgG1 antibodies. Interestingly, the vaccines supplemented with ginseng favoured IgG2. The vaccines used in the evaluations varied in their immunogenic potency. However, after the addition of ginseng the less immunogenic vaccine proved to be as potent as the better one without ginseng. Thus, the use of ginseng as a co-adjuvant provides a simple, safe and cheap alternative for improving the potency of aluminium hydroxide adjuvanted vaccines.

Adjuvants, Immunologic↗
PubMed2003

Tumor necrosis factor (TNF)alpha and its soluble receptor (sTNFR) p75 during acute human parvovirus B19 infection in children.

Human parvovirus B19 (HPV) has been shown to be involved in the pathogenesis of various connective tissue and autoimmune diseases. In order to gain more information on HPV possible role in these diseases, we have investigated some immune responses in patients with acute HPV infection, mainly the secretion of the proinflammatory cytokine tumor necrosis factor (TNF)alpha and it's antagonist--the soluble TNF receptor (sTNFR) p75. Thirteen children with acute HPV infection and 13 healthy volunteers were investigated for the presence of autoantibodies, lymphocyte subpopulation counts, levels of total immunoglobulins, IgG subclasses and complement. The levels of TNFalpha and sTNFR p75 were determined in serum and conditioned medium (CM) from unstimulated and LPS stimulated peripheral blood mononuclear cell (PBMC) cultures. There was no difference between patients and controls regarding autoantibodies, lymphocytes, immunoglobulins, IgG subclasses and complement. A significant imbalance between TNFalpha and sTNFR p75 was found in the patients group. TNFalpha concentrations were significantly higher both in sera and in CM from the patients as compared with the controls. The levels of sTNFR concentrations were either similar (in sera) or significantly lower (in CM) in the patients compared with the controls. The TNF index, representing the biologically available TNFalpha, was significantly higher in patient's sera and CMs. In view of these results, it is conceivable that infection with human HPV in otherwise healthy children may lead to a proinflammatory state. The presence of high levels of biologically available TNFalpha, in susceptible individuals, may in turn play a role in the pathogenesis of systemic autoimmune diseases in HPV infected individuals.

Acute Disease↗
PubMed2001

A quantitative, internally controlled real-time PCR Assay for the detection of parvovirus B19 DNA.

Parvovirus B19 is an erythrovirus causing diverse clinical manifestations ranging from asymptomatic or mild, to more severe outcomes in, for example, immune-compromised patients. B19 is spread primarily via the respiratory route, but it can also be transmitted via blood and blood products. Viral loads in blood or plasma donations amount up to 10(11) genome equivalents/ml. Therefore, screening of plasma for fractionation for the presence of B19 and removal of highly loaded donations is a way to limit considerably the input of B19 into production pools and to improve further the safety of plasma products. An assay for the quantitative detection of B19 DNA, based on real-time PCR using ABI Prism SDS7700 (TaqMan) is described here. This assay allows precise quantitation of viral loads over 7 orders of magnitude. An exogenous internal control (internal quality marker) is included in each individual sample to prevent false negative results. A linearized plasmid is used as an internal quality marker that contains the identical sequence of the B19 target sequence but with an altered probe hybridization site. This allows co-amplification of B19 and internal quality marker and co-detection of FAM (6-carboxyfluorescein) or VIC labeled probes respectively. The assay is validated according to current guidelines (of the International Conference on Harmonization, Paul Ehrlich Institute, and the Council of Europe) and is optimized for high throughput screening.

DNA, Viral↗
PubMed2003

Evaluation of anti-parvovirus B19 activity in sera by assay using quantitative polymerase chain reaction.

Human parvovirus B19 (B19) infects cells of erythroid lineage. Production of neutralizing antibodies (Abs) is indispensable for recovery from B19-related disease state. In this study, we used a convenient method to measure neutralizing activities in human sera by using a real-time quantitative PCR based assay. Erythroid cell line KU812Ep6 was incubated with test sera before infection with B19 virus. The copy number of B19-DNA in cultures was decreased in the presence of the sera from patients who recovered from acute B19 infection, whereas no decrease in B19-DNA was in cultures incubated with sera from healthy volunteers who had no B19 infection. The decrease in B19-DNA copy number was calculated and the inhibition percentage was expressed as neutralizing activity to B19. A clinical study showed that the levels of neutralizing ability were high in patients who recovered soon after acute B19 infection, but were low in some patients with a prolonged clinical course for recovery from B19 infection. This method is simple and convenient compared with methods described previously, showing its usefulness to evaluate the neutralizing activity to B19.

Animals↗
PubMed1998

Antigenic characterization of canine parvovirus strains isolated in Italy.

28 isolates of canine parvovirus type-2 (CPV-2) were obtained from dogs with hemorrhagic gastroenteritis in Italy. The antigenic structure of CPV-2 isolates was characterized, using four discriminating monoclonal antibodies. In addition, four vaccinal strains were examined. Similar to reports from Australia and the United Kingdom, a much higher prevalence of CPV-2a (25/28 isolates) was observed than the other variant type, CPV-2b (3/28 isolates). DNA fragments (2.2 kbp) of representative strains of CPV-2, CPV-2a and CPV-2b were amplified by the polymerase chain reaction (PCR) and the products were digested by the restriction enzymes (RE) RsaI, HpaII, HindIII and PvuII. The RvaI enzyme allows the differentiation of CPV-2 from CPV-2a and CPV-2b.

Animals↗
PubMed1999

Fluorescent and electron-microscopy immunoassays employing polyclonal and monoclonal antibodies for detection of goose parvovirus infection.

Polyclonal antibodies (PAbs) raised in geese and eight mice hybridomas secreting monoclonal antibodies (MAbs) against the goose parvovirus (GPV) were prepared. They were used for development of immunofluorescence (IF) and immunoelectron-microscopic (IEM) techniques to demonstrate the GPV infection in infected organs and biological fluids. The GPV antigens were established by immunofluorescence within the nuclei and the cytoplasm of many infected cells of the chorioallantoic membrane of goose and Peckin duck embryos, liver and heart of mortally diseased goslings. By means of IEM it was possible to detect the GPV in native organ homogenate supernatants and allantoic fluids. All techniques used in the study could be successfully applied for rapid diagnosis of the GPV infection. The test systems on the basis of MAbs should, however, be preferred. By means of immunoblotting (IB) using PAbs and MAbs four viral proteins (VP) with MW 88, 77, 65 and 60 kDa were demonstrated. Contrary to the others the VP with MW 65 kDa was the most antigenically reactive though invisible in the SDS-PAGE and Coomassie-blue dye-stained preparations.

Animals↗
PubMed1999

Detection of porcine parvovirus DNA by the polymerase chain reaction assay using primers to the highly conserved nonstructural protein gene, NS-1.

Porcine parvovirus (PPV) infection is associated with reproductive losses in swine and its causative agent, the PPV, has been isolated worldwide. Serological surveys and virus isolation studies throughout Brazil confirm the occurrence of PPV infection in this country. The most common methods to detect PPV infection are fluorescent antibody staining of fetal tissues, hemagglutination assay of tissue extracts and virus isolation from fetal tissues. Non-specificity and low sensitivity are the major drawbacks of these techniques. The development of a polymerase chain reaction (PCR) and nested-PCR assays for PPV DNA detection from infected cell lines and clinical samples is described. The primers were designed to a highly conserved region of the PPV genome which codes for the non-structural protein, NS-1. Results showed that PCR could detect PPV in titres at least 10(6) higher than the hemagglutination assay. The PCR and nested-PCR assays were used to detect successfully PPV DNA in clinical samples.

Abortion, Veterinary↗
PubMed1999

Chemiluminescence Western blot assay for the detection of immunity against parvovirus B19 VP1 and VP2 linear epitopes using a videocamera based luminograph.

A chemiluminescent Western blot (WB) assay was developed to detect the immune status against linear epitopes of parvovirus B19 structural proteins VP1 and VP2. The chemiluminescent WB assay combined the sensitivity of chemiluminescent substrates and the objective evaluation of the luminescent signal obtained with a new system which consists of a videocamera-based, high-performance, low light level imaging luminograph connected to a personal computer for image analysis. The potential for diagnostic purposes was evaluated using reference serum samples and 75 clinical samples from patients with different clinical conditions and laboratory evaluations regarding B19 infection. The chemiluminescent Western blot assay provided reproducible results, an objective evaluation of the results and a semi-quantitative analysis of the presence of antibodies against VP1 and VP2 in human sera. The chemiluminescent Western blot assay proved more sensitive than the classic colourimetric Western blot assay.

Adolescent↗
PubMed1999

Infection of the erythroid cell line, KU812Ep6 with human parvovirus B19 and its application to titration of B19 infectivity.

A human parvovirus B19 (B19) infectivity assay was developed using the erythroid cell line, KU812Ep6. KU812Ep6 was cloned for high efficiency infection with B19 in vitro, in the presence of erythropoietin by a limiting dilution method from the parent cell line, KU812. B19 was effectively propagated in KU812Ep6 and was detected for B19 antigens, VP1 and VP2. The titers of B19 positive sera measured with KU812Ep6 cells were in the range of 10(6) to 10(8) TCID50 ml. This KU812Ep6 infectivity assay had a 10(3)-10(4.5) higher sensitivity than the colony forming unit-erythroid (CFU-e) injury assay. It was calculated that one TCID50 needed 10(3) B19 genome copies, judging from the infectivity assay and semi-quantitative PCR. The KU812Ep6 infectivity assay was also used to determine infectivity of B19 in vitro, and to evaluate inactivation, as well as clearance of the virus. The inactivation of B19 by heating was carried out and infectivity declined from 10(4) TCID50 ml to < 10 TCID50 ml (lower limit of detection) at 60 degrees C for 3 h or at 70 degrees C for 30 min, but only decreased to 10(2.5) TCID50 ml at 50 degrees C for 8 h.

Antigens, Surface↗
PubMed2002

Genetic analysis of canine parvovirus isolates (CPV-2) from dogs in Italy.

Genetic and antigenic properties of 62 field isolates of canine parvovirus (CPV-2) collected from 1994 to 2001 in Italy were investigated. Antigenic characterisation was conducted using specific monoclonal antibodies (Mabs). The VP1\VP2 gene was amplified by PCR and characterised with restriction endonucleases to detect the 297 and 265 variant. The VP2 gene of 16 isolates was sequenced and molecular genetic analysis was conducted. The antigenic type prevalent among our isolates is type 2a as well as the 297 variant, which is also prevalent in the rest of Europe. Only the 9.7% of the isolates have the T265P mutation. The VP2 sequences of CPV-2 isolates were very similar to recent Asian isolates. In the threefold spike of CPV-699 a coding change was detected in the 440 residue where threonine was substituted by alanine: the same mutation has been found in two Asian CPV-2 isolates from leopard cats [Virology 278 (2000) 13]. Phylogenetic analysis revealed that the Italian CPV-2 strains followed the same evolution as observed in other countries and they gave no indication of a separate lineage.

Animals↗
PubMed1998

Typing of European strains of parvovirus B19 by restriction endonuclease analyses and sequencing: identification of evolutionary lineages and evidence of recombination of markers from different lineages.

European isolates of parvovirus B19 were analyzed by restriction enzyme analysis of PCR products of the VP1/2 coding region and sequencing of the same amplified region, five cloned fragments from each PCR product. Two main groupings were found based on three perfectly linked point deviations. On the assumption that identical point deviations causing the various restriction patterns regardless of time and origin of virus isolation were unlikely to emerge independently in different evolutionary lineages, traits of evolutionary lineages were identified, suggesting a clonal population structure of global circulating B19 strains. However, combinations of markers from different evolutionary lineages were also found, particularly in a strain derived from an individual chronically infected with B19 for more than 7 years. As chronically infected individuals might be subject to superinfections due to contacts or possibly due to blood transfusions or the administration of gamma-globulin, it is suggested that coexistence of, and recombination between variants of B19 of different phylogenetic origin incidentally occur in such individuals.

Capsid↗
PubMed2002

[Parvovirus B19 outbreak in a rural community in Alicante].

BACKGROUND: Parvovirus B19 (PVB19) has been identified as the cause of erythema infectiosum. The epidemiology of PVB19 has not been extensively studied in Spain or in the autonomic community of Valencia. The aim of this work is to describe an outbreak of PVB19 infection occurring in the area of Monforte del Cid, Alicante. METHODS: A probable case was defined as: all subjects living in Monforte who presented a rash (mainly facial) and/or arthralgia starting from November 1999. A confirmed case was defined as: a probable case confirmed by laboratory analysis or a case having an epidemiological link. Laboratory confirmation included specific IgG or IgM antibodies to PVB19. Cases were mainly detected through the Monforte del Cid Primary Health Care Center. RESULTS: The outbreak occurred from November 1999 to August 2000. A total of 118 cases were detected, giving an overall attack rate (AR) of 23.2 cases per 1,000 inhabitants. The highest rates were in the age groups of 0-4 years old (AR 5 114.5 per 1,000) and 5-9 years old (AR 5 180.3 per 1,000). By gender, the AR per 1,000 inhabitants was 26.9 in men and 16.7 in women. Two of the cases were pregnant women and one of them had a miscarriage. CONCLUSIONS: The outbreak of erythema infectiosum lasted 10 months and mainly affected children under 14 years old. Active surveillance was focussed on women in the first three months of pregnancy.

Abortion, Spontaneous↗
PubMed1993

[Periarteritis nodosa and parvovirus B19 infection].

The authors report a case of polyarteritis nodosa which coincide with serological conversion to parvovirus B19. After review of the literature, they recall the role of this virus in human pathology and also the role of different infectious agents in the PAN pathogenesis.

Aged↗
PubMed2000

Mapping of B-cell epitopes on human parvovirus B19 non-structural and structural proteins.

In the present study, the immune reactivity to Parvovirus B19 (B19) proteins and variations in antigenic reactivity in different clinical manifestations were investigated. Sera from healthy B19 IgG positive individuals were evaluated for antibody reactivity against linear peptides. Three antigenic regions (amino acid number 191-206, 271-286, 371-386) on the B19 non-structural (NS) protein 1 were identified. The highest seroreactivity against these peptides was found against amino acid number 271-286. Seroreactivity in this group of individuals was also investigated against peptides representing selected neutralising regions of the B19 capsid proteins viral protein (VP) 1/VP2. The antigenic NS1 and VP1/VP2 regions, thus defined, were further mapped by seroreactivity against peptides containing specific deletions. The frequencies of seroreactivity against the NS1 and VP1/VP2 peptides in healthy B19 IgG positive individuals were similar to those in HIV-seropositive and persistently B19 infected patients, except that the latter group showed a lower reactivity to the C-terminal end of VP1/VP2. The identification of antigenic regions and corresponding seroreactivity in asymptomatic and persistently B19-infected patients is important for the understanding of B19-pathogenesis and for the development of B19 vaccine candidates.

Amino Acid Sequence↗
PubMed2000

Effect of different baculovirus inactivation procedures on the integrity and immunogenicity of porcine parvovirus-like particles.

We have demonstrated earlier the usefulness of recombinant porcine parvovirus (PPV) virus-like particles (VLPs) as an efficient recombinant vaccine for PPV. Here, we have demonstrated that preparations of PPV VLPs could be contaminated by recombinant baculoviruses. Since these baculoviruses can be a problem for the registration and safety requirements of the recombinant vaccine, we have tested different baculovirus inactivation strategies, studying simultaneously the integrity and immunogenicity of the VLPs. These methods were pasteurization, treatment with detergents and alkylation with binary ethylenimine (BEI). The structural and functional integrity of the PPV VLPs after the inactivation treatments were analyzed by electron microscopy, hemagglutination, double antibody sandwich (DAS)-ELISA and immunogenicity studies. Binary ethylenimine and Triton X-100 inactivated particles maintained all the original structural and antigenic properties. In addition, PPV VLPs were subjected to size-exclusion chromatography to analyze the presence of VP2 monomers or any other contaminant. The resulting highly purified material was used as the standard of reference to quantify PPV VLPs in order to determine the dose of vaccine by DAS-ELISA. After immunization experiments in guinea pigs, the antibody titers obtained with all the inactivation procedures were very similar. Triton X-100 treatment was selected for further testing in animals because of the speed, simplicity and safety of the overall procedure.

Animals↗
PubMed2001

Inactivated recombinant plant virus protects dogs from a lethal challenge with canine parvovirus.

A vaccine based upon a recombinant plant virus (CPMV-PARVO1), displaying a peptide derived from the VP2 capsid protein of canine parvovirus (CPV), has previously been described. To date, studies with the vaccine have utilized viable plant chimaeric particles (CVPs). In this study, CPMV-PARVO1 was inactivated by UV treatment to remove the possibility of replication of the recombinant plant virus in a plant host after manufacture of the vaccine. We show that the inactivated CVP is able to protect dogs from a lethal challenge with CPV following parenteral immunization with the vaccine. Dogs immunized with the inactivated CPMV-PARVO1 in adjuvant displayed no clinical signs of disease and shedding of CPV in faeces was limited following CPV challenge. All immunized dogs elicited high titres of peptide-specific antibody, which neutralized CPV in vitro. Levels of protection, virus shedding and VP2-specific antibody were comparable to those seen in dogs immunized with the same VP2- peptide coupled to keyhole limpet hemocyanin (KLH). Since plant virus-derived vaccines have the potential for cost-effective manufacture and are not known to replicate in mammalian cells, they represent a viable alternative to current replicating vaccine vectors for development of both human and veterinary vaccines.

Amino Acid Sequence↗
PubMed2003

Effect of priming/booster immunisation protocols on immune response to canine parvovirus peptide induced by vaccination with a chimaeric plant virus construct.

Expression of a 17-mer peptide sequence from canine parvovirus expressed on cowpea mosaic virus (CPMV) to form chimaeric virus particles (CVPs) creates vaccine antigens that elicit strong anti-peptide immune responses in mice. Systemic (subcutaneous, s.c.) immunisation and boosting with such CVP constructs produces IgG(2a) serum antibody responses, while mucosal (intranasal, i.n.) immunisation and boosting elicits intestinal IgA responses. Combinations of systemic and mucosal routes for priming and boosting immunisations were used to examine their influence on the level, type and location of immune response generated to one of these constructs (CVP-1). In all cases, s.c. administration, whether for immunisation or boosting, generated a Th1-biased response, reflected in a predominantly IgG(2a) serum antibody isotype and secretion of IFN-gamma from in vitro-stimulated lymphocytes. Serum antibody responses were greatest in animals primed and boosted subcutaneously, and least in mucosally vaccinated mice. The i.n. exposure also led to IFN-gamma release from in vitro-stimulated cells, but serum IgG(2a) was significantly elevated only in mice primed intranasally and boosted subcutaneously. Peptide- and wild-type CPMV-specific IgA responses in gut lavage fluid were greatest in animals exposed mucosally and least in those primed and boosted subcutaneously or primed subcutaneously and boosted orally. Lymphocytes from immunised mice proliferated in response to in vitro stimulation with CPMV but not with peptide. The predominant secretion of IFN-gamma from all immunising/boosting combinations indicates that the route of vaccination and challenge does not alter the Th1 bias of the response to CVP constructs. However, optimal serum and intestinal antibody responses were achieved by combining s.c. and i.n. administration.

Amino Acid Sequence↗
PubMed1997

Immunogenicity of a low-passage, high-titer modified live canine parvovirus vaccine in pups with maternally derived antibodies.

The study evaluated the ability of a low-passage, high-titer modified live canine parvovirus (CPV) vaccine to produce seroconversion in pups with maternally derived hemagglutination inhibition (HI) titers ranging from < 8 to < or = 256. The vaccine's low-passage CPV strain was less attenuated and therefore more infective than conventional modified live CPV strains in order to overcome relatively greater levels of maternally derived antibodies, the principal cause of CPV vaccine failures in pups. To assess vaccine performance under field conditions, healthy pups presented at five private veterinary clinics were used as test animals. A single dose of vaccine was given to 59 pups at 12 weeks of age (Group A). To accommodate the protocol of clinics where earlier CPV vaccination was practiced, 87 other pups were vaccinated with two doses, the first at 8-10 weeks of age, and the second at 12 weeks of age (Group B). Geometric mean HI titers were measured for blood samples obtained at the time of vaccination and at 14 weeks of age. Seroconversion was considered to have occurred if pups developed a fourfold or greater increase in HI titer to a level > or = 64. Of the 59 pups in Group A, 100% seroconverted following the single vaccine dose at 12 weeks of age. Of the 87 Group B pups, 82 (94.3%) seroconverted following either of the two vaccine doses. A geometric mean HI titer of 4828 was measured for Group A, and a geometric mean HI titer of 2028 was measured for Group B. An overall seroconversion rate of 96.5% was achieved in pups with maternally derived HI titers < or = 256.

Animals↗