G. Gentilomi

Active or recent parvovirus B19 infection in children with Kawasaki disease

Because parvovirus B19 occasionally causes some of the features typical of Kawasaki disease, we investigated B19 involvement in 15 children with Kawasaki disease. Active or recent B19 infection, as shown by B19-DNAaemia, positive B19-specific IgM antibodies, or both, was diagnosed in 10 patients (67%). A high frequency of all major criteria for diagnosis of Kawasaki disease (60%), anaemia (60%), coronary aneurysms (30%), and arthropathy (30%) was found in children with B19-associated Kawasaki disease. Thus B19 may have a pathogenic role in the development of Kawasaki disease, with other predisposing factors.

Synthesis and antiviral assays of some 2-substituted benzimidazole-N-carbamates.

Some 2-substituted benzimidazole-N-carbamates were synthesized and tested in vitro for antiviral activity. Two derivatives were active at noncytotoxic concentrations. The results confirmed the importance of the substituents at the 2-position of benzimidazole; an isopropylcarboxamide group led to the best activity.

Identification of an immunodominant peptide in the parvovirus B19 VP1 unique region able to elicit a long-lasting immune response in humans.

The immune response against parvovirus B19 is mainly directed against the two structural proteins, VP1 and VP2. The amino terminal half of the VP1 unique region has been shown to elicit a dominant immune response in humans, more effective than other linear epitopes and also it has been seen to contain significant neutralizing linear epitopes. Three overlapping recombinant peptides corresponding to amino acids 2-40 (VP1-A), amino acids 32-71 (VP1-B), and amino acids 60-100 (VP1-C) of the VP1 unique region were produced by a procaryotic expression system. These peptides were used as antigens in a Western blot assay to detect specific immunoglobulin G (IgG) in serum samples from blood donors of different age groups with documented signs of a past B19 infection. Fragment VP1-C appeared significantly immunodominant over the other peptides, reacting with specific IgG in 86% of serum samples. The fragment VP1-C corresponds to a sequence with a known neutralizing activity and seems able to elicit a long-lasting immune response because specific IgG were present in blood donors of all age groups. VP1-C would therefore appear to be an attractive candidate as a component of a subunit vaccine.

IgG Response to the Immunoreactive Region of Parvovirus B19 Nonstructural Protein by Immunoblot Assay with a Recombinant Antigen

A total of 190 serum samples from patients with different clinical manifestations in whom B19 parvovirus infection was suspected and 108 control serum samples were analyzed for the IgG response against parvovirus B19 nonstructural protein. An immunoblot assay was developed using the immunoreactive region of the B19 nonstructural protein as recombinant antigen. Virologic (B19 DNA) and serologic (IgM and IgG anti-VP) markers of B19 infection were also investigated. There was an even distribution of IgG nonstructural antibodies in serum samples of parvovirus B19-infected patients and controls (27.7% and 21.7%, respectively) that were mainly associated with patterns of past B19 infection.

Extent of sequence variability in a genomic region coding for capsid proteins of B19 parvovirus

SummaryGenomic variability in human parvovirus B19 was analysed by direct partial sequencing of different isolates collected in Italy between 1989 and 1994. DNA was purified from viremic serum samples and the region of viral genome coding for structural proteins was amplified by polymerase chain reaction and partially sequenced (nt. 2400–3400). Data were compared to reference isolates Wi (U.K., 1973) and Au (U.S.A., 1982). The average relative distance between isolates was estimated at the low level of 0.61%, comparable with the distance to reference isolates. Out of 22 nucleotide substitutions found, 9 resulted in amino acid changes. From our results, this region of human parvovirus B19 genome appears to be stably conserved.

Efficient parvovirus B19 DNA purification and molecular cloning.

A simple and efficient method of purification and molecular cloning of Parvovirus B19 DNA directly from small quantities of viremic sera was developed. Purified virions were lysed in annealing conditions, then viral DNA purification in double strand (ds) DNA form was achieved using an affinity DNA binding matrix. Affinity purification yielded a consistently high recovery of viral DNA. Using affinity purified ds viral DNA, we efficiently and stably cloned the complete coding internal unique sequence of B19 DNA. In our cloning strategy AatII and BamHI restriction endonuclease sites were exploited. This permitted cleavage of the 5.0 kbp AatII fragment in two AatII-BamHI fragments which could be efficiently cloned in a directional way in pUC18 plasmid vector. The availability of the two cloned AatII-BamHI fragments thus allowed the construction of a full length clone in a single ligation reaction.

A hybrido-immunocytochemical assay for the in situ detection of cytomegalovirus DNA using digoxigenin-labeled probes.

A non-radioactive hybrido-immunocytochemical assay for the detection of cytomegalovirus (CMV) DNA in infected cells was developed. Two different DNA fragments belonging to the repeated sequences of CMV genome were used to construct the hybridization probe. The probe was constructed by incorporating deoxyuridine triphosphate labeled with digoxigenin. The in situ hybridized CMV DNA probe was immunocytochemically visualized by anti-digoxigenin. Fab fragments labeled with alkaline phosphatase. This procedure permitted the DNA detection, in the nuclei of infected cells fixed at 48 h after infection, of the Towne CMV reference strain and 21 different laboratory-isolated CMV strains. Our assay demonstrated a high specificity, sensitivity and reproducibility.

Automated detection of digoxigenin-labelled B19 parvovirus amplicons by a capture hybridization assay

An automated method to identify B19 amplicons, directly labelled with digoxigenin during amplification reaction was developed. The labelled amplicons were hybridized with a biotinylated B19 oligo-probe and captured on commercially available test tubes coated with streptavidin. The hybridized amplicons labelled with digoxigenin were detected using anti-digoxigenin Fab fragments conjugated to peroxidase and the colourimetric reaction automatically evaluated as an immunoenzymatic assay. Fifty serum samples were tested by the assay and the results were in accordance with those obtained by Southern blot analysis of amplified products. Due to the high sensitivity, specificity and reproducibility shown, the assay seems to be a practical and reliable test for the diagnosis of B19 infection and can be easily adapted to identify any digoxigenin-labelled amplified product of viral genomes.

Double In Situ Hybridization for Detection of Herpes Simplex Virus and Cytomegalovirus DNA Using Non-radioactive Probes'

We describe a double in situ hybridization assay for the simultaneous detection of Herpes simplex virus (HSV) and cytomegalovirus (CMV) DNA in infected cell cultures using non-radioactive-labeled probes. This work used a biotinylated HSV DNA probe, which can be revealed by an avidin-biotin-peroxidase complex and a digoxigenin-labeled CMV DNA probe, visualized by anti-digoxigenin F(ab) fragments conjugated with alkaline phosphatase. Light microscopy visualization was achieved by the contrasting colors of appropriate peroxidase and alkaline phosphatase reaction products (red and dark blue, respectively). The time required to perform the double hybridization assay was about 3 hr. This double hybridization assay proved to be sensitive, specific, and provided good resolving power.

Synthesis and antiviral assays of some benzimidazole nucleosides and acyclonucleosides

Some benzimidazole nucleosides and acyclonucleosides were synthesized and tested in vitro as antiviral agents. None of them showed significant activity. Replacement of the benzenesulphonyl group at N-1 with the ribofuranosyl moiety or with the acyclovir side-chain was deleterious.