Prevalence of nucleic acid sequences specific for human parvoviruses, hepatitis A and hepatitis E viruses in coagulation factor concentrates.

BACKGROUND AND OBJECTIVES: Due to their high resistance to inactivation procedures, nonenveloped viruses such as parvovirus B19, human bocavirus (HBoV), human parvovirus 4 (PARV4), hepatitis A (HAV) and hepatitis E virus (HEV) pose a particular threat to blood products. Virus transmission to patients treated with blood products presents an additional burden to disease. We determined the frequency and the amount of nucleic acid specific for nonenveloped viruses in recently manufactured preparations of commercial coagulation factor concentrates. MATERIALS AND METHODS: At least three different batches of each of 13 different plasma-derived and recombinant coagulation factor products were tested for the presence and the amount of nucleic acid for parvovirus B19, HBoV, human parvovirus 4, hepatitis A virus and HEV by using quantitative polymerase chain reaction. RESULTS: Whereas none of the recombinant products tested positive for any of these viruses, parvovirus B19 DNA with amounts ranging between 2×10(1) and 1.3×10(3) genome equivalents/ml was detected in five plasma-derived products. In addition to parvovirus B19 genotype 1, genotypes 2 and 3 were observed in two batches of a factor VIII/von-Willebrand factor product. In two products (one factor VIII concentrate and one activated prothrombin complex concentrate), a combination of both genotypes 1 and 2 of parvovirus B19 was detected. CONCLUSION: The data show that nucleic acids from several relevant nonenveloped viruses are not found at detectable levels in coagulation factor concentrates. In some cases, parvovirus B19 DNA was detectable at low levels. Testing of the plasma pools for the full range of parvovirus genotypes is advocated for ensuring product safety.

Persistent parvovirus B19 infection detected by specific CD4+ T-cell responses in a patient with hepatitis and polyarthritis.

We, here, report the case of a parvovirus B19 infection in an immunocompetent male patient presenting with acute hepatitis and polyarthritis. To follow the course of infection, we used a previously established enzyme-linked immunosorbent spot assay (ELISPOT) technique to detect CD4+ T cells specific for viral proteins. Even though symptoms of arthritis and hepatitis resolved in the immunocompetent individual within a few weeks, viral DNA in serum and CD4+ T cells specific for the viral protein VP1 unique region were still detectable more than 6 month after the onset of symptoms, thus pointing to a persistent state of infection. On the basis of this observation, we hypothesize that the intensity of liver involvement correlates with the likelihood of developing persistent parvovirus B19 infection. The described ELISPOT technique to detect virus-specific CD4+ T cells provides an excellent tool to analyse the state of parvovirus B19 infection for future studies to test this hypothesis.

Parvovirus B19: the causative agent of dilated cardiomyopathy or a harmless passenger of the human myocard?

Parvovirus B19 infections may cause a widespread benign and self-limiting disease in children and adults known as erythema infectiosum (fifth disease). Several further manifestations are associated with B19 infections, such as arthralgias, arthritis, leucopenia and thrombocytopenia, anaemia and vasculitis and spontaneous abortion and hydrops fetalis in pregnant women. Persistent infections with continuous virus production may occur in immunocompetent as well as in immunosuppressed individuals. Parvovirus B19 infections have been frequently implicated as a cause or trigger of various forms of autoimmune diseases affecting joints, connective tissue and large and small vessels. Autoimmune neutropenia, thrombocytopenia and haemolytic anaemia are known as sequelae of B19 infections. The molecular basis of the autoimmune phenomena is unclear. Many patients with these long-lasting symptoms are not capable of eliminating the virus or controlling its propagation. Furthermore, latent viral genomes have been detected in cells of various organs and tissues by PCR. At present, it is not clear if these cells produce viral proteins and/or infectious B19 particles, if the virus genome can be reactivated to productive replication and if the presence of viral DNA indicates a causative role of parvovirus B19 with distinct diseases.

Immunoprecipitation as a tool for studying humoral immunity of natural and experimental hosts of Herpesvirus saimiri.

Herpesvirus saimiri strain 11 and attenuated H. saimiri strain 11 proteins synthesized during the lytic cycle of virus replication were used for immunoprecipitation with various sera from natural (Saimiri sciureus) and experimental (Saguinus nigricollis, Saguinus fuscicollis, Aotus trivirgatus, New Zealand White rabbits) hosts. The analysis of the precipitates separated in sodium dodecyl sulfate:polyacrylamide gels revealed that in tumor-developing animals a specific set of viral polypeptides were precipitated, which were not precipitated by sera obtained from the natural host Saimiri sciureus. Using labeled proteins from H. saimiri 11 and its attenuated strain, respectively, a difference was shown after precipitation with a serum raised against infected cell proteins of H. saimiri 11.

"Contact voltage" in nanoparticle/molecule connections.

This work presents conclusive evidence that connecting Pt and Co nanoparticles stabilized by an aluminum-organic shell with molecular spacers interacting with this shell can induce notable changes in the electronic structure of the metal. X-ray absorption spectroscopy measurements at the Al K-, the Pt L(III)-, and the Co K-edge provide consistent evidence for this effect. The changes induced by cross-linking with an acidic spacer are discussed in detail as an example to elucidate the mechanism of this effect. It turns out that a reconfiguration of the protection shell that occurs upon networking is responsible for the observed changes.

A common epitope on major allergens from non-biting midges (Chironomidae).

A synthetic peptide corresponding to sequence 91-101 of the Chironomus thummi thummi haemoglobins (Chi t I) components III and IV was used to investigate binding and cross-reactivity with polyclonal human IgE and rabbit IgG antibodies and murine IgG1 subclass monoclonal antibodies (MABs). The synthetic peptide reacted with antibodies from all three mammals. The specificity of the reaction, especially that with IgE antibodies was shown by dose dependent inhibition with native Chi t I component III. Epitope(s) reacting with these antibodies were also found in haemoglobins from 14 of the 15 chironomid species analyzed. The synthetic peptide III/IV 91-101 enabled the identification of an important antigenic/allergenic determinant of the broadly distributed insect family Chironomidae. The antigenic potency of this synthetic peptide as shown by testing with human IgE, rabbit IgG and mouse MABs, and the widespread occurrence of the epitope in an identical or homologous sequence and/or superficial location, qualifies the peptide for therapeutic applications in medicine.

Carrier bound synthetic oligopeptides in ELISA test systems for distinction between HIV-1 and HIV-2 infection.

A series of synthetic carrier bound oligopeptides derived from corresponding regions of the core and envelope proteins of HIV-1 and HIV-2 were used in enzyme-linked immunoabsorbent assays (ELISA) for serodiagnosis of HIV-1 and HIV-2 infected individuals. The combination of peptides from regions either conserved or highly variable between the two virus types allowed the identification of HIV infection in general and the differentiation between HIV-1 and HIV-2. No specific reaction was found in seronegative individuals. The use of peptides bound to the same polystyrene carrier as in peptide synthesis allowed the establishment of a highly specific and sensitive test system without the risk of unspecific cross-reaction due to contamination with bacterial or cellular protein material.

Use of synthetic oligopeptides in identification and characterization of immunological functions in the amino acid sequence of the envelope protein of HIV-1.

Following computer-assisted analysis of the amino acid sequence of various HIV-1 isolates, we synthesized a series of oligopeptides derived from variable and conserved regions of the envelope protein complex gp120/gp41. The peptides were used in ELISA tests for their reactivity with human antisera from HIV-1 positive individuals; patients with clinically manifested AIDS showed only a rather limited reaction, predominantly with two peptides (p102-112, p316-326), which is in contrast to sera from HIV-1 positive asymptomatic individuals, whose sera were reactive with almost all peptides. Using consecutive sera of the same patients, decreasing antibody titers to defined epitopes could be shown to occur during the development of AIDS. Cellular immune response recognition was analyzed in T-cell proliferation assays by [3H]thymidine incorporation. One peptide localized in a conserved region clearly induced proliferation of T-cells. Those data were combined to a map of the functions localized in the various regions of the HIV-1 envelope proteins.

HIV-1 peptides induce a proliferative response in lymphocytes from infected persons.

In a comprehensive search for T-cell epitopes of HIV-1, several new regions were discovered. The analysis was performed with lymphocytes of HIV-1-infected persons in various stages of the infection. Peptides covering the entire group antigen (gag), and transmembrane (gp41) regions, and one-half of envelope (gp120) regions of HTLV-IIIB were studied. Both common and patient-unique responses were identified. Twelve common gag T-cell sites were discovered, as well as patient-unique activating peptides. The gag peptides elicited the most frequent cell responses and the responses remained in late stages of disease. Only 1 of the 12 T-cell activating gag peptides was reactive with specific anti-HIV IgG. Eighteen common env-representing peptides evoked T-cell responses. They could be grouped into four previously undescribed regions of gp120 and two known sites, the hypervariable stretch and part of the CD4 binding region. Cell responses to env peptides were common in early stages of disease and tended to decrease in ARC and AIDS. The gp41-representing peptides evoked a cellular response to a region close to the N-terminus of the hydrophobic transmembrane region. In addition to the T-cell activating peptides, peptides representing p15 and p19 as well as previously recognized regions of gp120 and gp41 appeared to be potent B-cell epitopes.

Carrier-bound synthetic peptides. Use as antigen in HIV-1 ELISA tests and in antiserum production.

Chemically synthesize carrier-bound peptides have been used as antigens in diagnostic test systems (ELISA) and for raising antipeptide-specific antisera. The method does not require prior cleavage of the peptides from the support used for the solid-phase synthesis. Using the same resin for both the synthesis and the subsequent applications it was possible to avoid expensive and time-consuming purification procedures and artificial recoupling to solid supports. A quick and specific ELISA-based diagnostic test system for HIV-specific antipeptide antibodies in human sera was established. In addition the carrier-bound peptides were shown to be potent antigens for raising antibodies in animals.

Inhibition of cell adhesion to the virus by synthetic peptides of fiber knob of human adenovirus serotypes 2 and 3 and virus neutralisation by anti-peptide antibodies.

The fiber knob of adenovirus (Ad) causes the first step in the interaction of adenovirus with cell membrane receptors. To obtain information on the receptor binding site(s) several synthetic peptides derived from Ad2 and Ad3 fiber head sequences and their antisera were tested for interference with virus attachment to HeLa and FL cells and cell adhesion to viruses. The anti-peptide sera were also evaluated in ELISA and virus neutralisation test. Ad2 (of subgroup C) and Ad3 (of subgroup B) attachment was not significantly inhibited by peptides corresponding to the amino acid residues 535-554, 555-573, 562-582 of Ad2 fiber or 210-225, 267-283, 291-306 and 300-319 of Ad3 fiber. However, microplate pre-adsorbed Ad3 fiber residues 210-225 and 267-283 could bind FL and HeLa cells, and 1 mg/ml of Ad3 fiber residues 267-283 inhibited the cell adhesion to Ad3 virus to approximately 90%. This peptide may participate in the receptor binding site of Ad3 fiber. ELISA reactive anti-peptide antibodies against the homologous peptide and virus did not significantly reduce the cell adhesion to the immobilised virus or the virus attachment to cells, but in the neutralisation assay antibodies raised to Ad2 fiber residues 555-573 and 562-582 and Ad3 fiber residues 210-225 caused neutralisation of the homologous virus at serum dilutions of 1:500 and 1:32, respectively. The corresponding peptides and one further peptide of Ad2 fiber and two of Ad3 fiber seem to contain neutralisation epitopes.

Flow-cytometric determination of peptide-class I complex formation. Identification of p53 peptides that bind to HLA-A2.

A novel class I-peptide-binding assay was developed and used to identify a series of peptides derived from the human p53 tumor-suppressor gene product capable of binding the HLA-A2 class I allele. Brief pH 3.3 acid treatment of human cell lines rapidly denatures pre-existing class I complexes, as detected by loss of binding of conformation-dependent mAbs, leaving only free class I heavy chains associated with the viable cell surface. These heavy chains may be induced to refold and be recognized by antibodies (in 2-4 hours) when acid-treated cells are coincubated with exogenous beta 2-microglobulin and peptides capable of binding the relevant class I allele examined. This assay, with a detection limit of 1-10 nM peptide, was used to screen the capacity of a panel of nine peptides bearing HLA-A2-binding motifs and derived from the human p53 tumor-suppressor protein sequence. Eight of the nine peptides bound to, and reconstituted, HLA-A2 on acid-treated cells. This assay system will enable the rapid identification of peptides binding to any class I allele, which is the initial prerequisite for elucidating potential CD8+ T-cell epitopes.

Reactivities of HIV-1 gag-derived peptides with antibodies of HIV-1-infected and uninfected humans.

A group of 41 peptides, each 24 amino acids long and overlapping with each other by 12 residues spanning the total gag open reading frame (orf) of HIV-1 (HTLV-IIIBH 10 isolate) were synthesized using Fmoc chemistry. The purified compounds were used in ELISA assays and tested for antibody reactivities in sera of human HIV-1-infected and noninfected individuals. Sera of HIV- humans showed reactivity against four defined regions, two in p17, one in p24, and one in p15. The values of these reactivities were elevated especially in serum samples of HIV- individuals showing cross-reaction with gag proteins on Western blot. Amino acid sequence comparison of HIV-1 gag proteins with those of human endogenous retroviruses (ERV K10, ERV 3) revealed significant similarities predominantly in the domains showing elevated antibody cross-reactions. The majority of sera from HIV-1+ individuals showed strong reactivities to the cross-reactive regions and to various other peptide sequences, a sequential epitope recognized by all HIV-1+ sera could, however, not be identified. The results suggest that human individuals may have immune reactions to endogenous retroviral protein sequences, which are enhanced by infections with HIV-1. Specific antibodies to HIV-1 gag proteins are probably mainly directed to tertiary structure defined epitopes formed by particle formation of the p24 monomers to the nucleocapsid.

Acute parvovirus B19 infection in connection with a flare of systemic lupus erythematodes in a female patient.

BACKGROUND: Since its discovery parvovirus B19-infections could be linked to a growing variety of diseases. Besides the harmless exanthema erythema infectiosum perferentially observed with B19-infections in childhood a panel of rather serious and also chronic courses that may be associated with anemia, thrombocytopenia, arthritis and others have been described. OBJECTIVE: In a 26-year-old female patient an acute parvovirus B19-infection was followed by a serious episode of systemic lupus erythematosus (SLE). Here we demonstrate the clinical and serological parameters which were observed in the patient during that episode in addition to the nucleotide sequence of the virus isolate. RESULTS AND CONCLUSION: In this patient parvovirus B19 was not the initial causative agent for SLE. However the B19 infection was followed by a severe flare of SLE and therefore may be considered as an enhancer of the autoimmune disease. The amount of nucleotide variability observed in the viral genome was in the range known from other B19 isolates. An elevated degree of mutations in antigenic domains was not detectable. Therefore, we would like to emphasize the possible role of parvovirus B19 in the aetiology or the enhancement of autoimmune diseases like SLE and the necessity of an according differential diagnosis.

Chronic autoimmune thrombopenia/neutropenia in a boy with persistent parvovirus B19 infection.

OBJECTIVE: We report an 11-year-old boy presenting with splenomegaly, chronic thrombocytopenia and concordant neutropenia. RESULTS: In contrast to autoantibodies against platelets, there were no detectable neutrophil-specific autoantibodies present in this patient. Extensive serologic investigations revealed increased IgM- and IgG-antibody titers against parvovirus B19. A nested polymerase chain reaction (PCR) showed parvovirus B19-specific sequences in the patient's bone-marrow cells but not in the serum. Specific antibodies against the structural proteins VP1 and VP2 in addition to those against non-structural protein NS1 of parvovirus B19 were detected by Western blot analysis. Thrombocytopenia and neutropenia responded to immunosuppressive therapy and subsequent splenectomy, the latter being necessary due to severe side-effects of steroid medication. CONCLUSION: Autoimmune thrombocytopenia/neutropenia may have been triggered and/or sustained by a chronic parvovirus B19 infection. Patients with this very rare disorder should be screened for this virus.

Monoclonal antibodies directed against tick-borne encephalitis virus with neutralizing activity in vivo.

Monoclonal antibodies (MoAbs) were raised against the tick-borne encephalitis (TBE) virus, strain K23. The reactivities of 14 selected MoAbs were characterized by ELISA, Western blot analysis, haemagglutination inhibition, immunoprecipitation, in vivo protection and in vitro neutralization tests. All MoAbs reacted only with the glycoprotein E. The binding epitope of one MoAb could be delimited by a synthetic peptide to amino acids 306-339 representing one immunodominant loop structure of the glycoprotein E. The MoAbs exhibited individual reactivities against 13 different TBE virus isolates in ELISA and immunoblot test ranging from type-specific reactions to a broad reactivity with all isolates. Four MoAbs also showed a cross-reaction with other flaviviruses like West Nile virus and/or Yellow fever virus in immunoblot analysis. By competition ELISA the MoAbs could be divided into five different reaction patterns. Four MoAbs showed neutralizing activity with titers in the range 1:140 to 1:5,000 in an in vitro assay. These neutralizing activities could be confirmed by an in vivo mouse challenge model. The MoAbs are useful for diagnostic purposes and for differentiation of TBE virus strains and other flaviviruses.

Seroepidemiology of human bocavirus in Apulia, Italy.

A serological survey was performed to determine the prevalence of antibodies against human bocavirus in an Apulian population. Anti-hBoV IgG antibodies were analysed in 1206 inhabitants (age range, 1month-84years) using a standardized ELISA test based on the use of recombinant hBoV VP2 virus-like particles. In total, 1075 (89.1%) of 1206 participants (mean age 32±24.8years) displayed anti-hBoV-IgG. The seroprevalence increased significantly (p<0.0001) in children from 2-4years (64.2%) to 5-9years (96.4%). A similar trend was observed in both male and female subjects. In conclusion, our results show that hBoV infection is common in this population, especially in children.

Impact of parvovirus B19 infection on paediatric patients with haematological and/or oncological disorders.

To determine the frequency and the impact of parvovirus B19 (B19V) infection and its influence on the course of haematological and/or oncological diseases in paediatric patients, consecutive serum and bone marrow samples from 110 were analyzed for markers of acute, past and persistent B19V-infection using qPCR, ELISA and WesternLine. Twenty-seven out of 110 (24.5%) children suffered from non-malignant diseases (anaemia, pancytopenia, autoimmune disorders); 68/110 (61.8%) patients had developed leukaemia, malignant lymphoma or solid malignant tumours; 15/110 patients (13.6%) presented with other symptoms. At admission, B19V-specific IgM and IgG indicating acute or previous B19V-infection were observed in 5 (4.5%) and 48 patients (43.6%), respectively. B19V-DNA (10(3) -10(9) geq/mL) was detectable in serum and/or bone marrow of 22 patients (20.0%). These suffered from leukaemia (5), non-Hodgkin lymphoma (2), solid tumours (6), autoimmune (4) and haematological (4) disease and fever (1). During clinical observation four further leukaemia patients developed viraemia and persistent B19V-infection was observed in 13/22 DNA-positive patients. Treatment of B19V-DNA-positive cancer patients was associated with more supportive therapy involving erythrocyte and thrombocyte transfusion and/or antibiotic therapy. Acute B19V-infection has been frequently observed in paediatric patients with haematological and/or oncological disease. In patients with non-malignant diseases anaemia or autoimmune disorders were diagnosed in association with B19V-infection. Furthermore, a significant number of cancer patients displayed markers for acute, recent or persistent B19V-infection. This association may be strengthened by frequent treatment with blood products combined with therapeutic immune suppression. In B19V-infected cancer patients supportive therapy was more complex.