Werner Seidel

Rapid identification of subgenera of human adenovirus by serological and PCR assays

Bacterially expressed recombinant protein IX (pIX) of human adenovirus serotype 2 (Ad2) and 3 (Ad3) was evaluated for use as a subgenus-specific antigen by enzyme-linked immunosorbent assay (ELISA) and Western blotting. Patients sera positive by ELISA for the genus-specific adenovirus hexon antigen recognized the recombinant pIX of Ad2 and Ad3 in a subgenus-specific manner by both assays. Polyclonal rabbit serum raised against the recombinant Ad2pIX reacted strongly by indirect immunofluorescence assay, with Adl, 2 and 5 (subgenus C) but not with serotypes representing other subgenera. In a similar way, anti-Ad3pIX reacted with Ad3, 7, 11 and 14 (subgenus B), but not with serotypes representing other subgenera. A polymerase chain reaction showed that the complete pIX gene could be amplified in a subgenus specific fashion using primers specific for Ad3 (subgenus B), Ad2 (subgenus C), or Ad40/41 (subgenus F). The pIX gene from the available isolates of subgenus A, D and E was not amplified with these primers. The use of pIX-based serological assays is useful for subgenotyping as a primary screen of anti-Ad sera. It is much more rapid than the currently used neutralization assay or hemagglutination inhibition test. The application of anti-pIX sera by immunofluorescence and a pIX gene-based PCR are rapid methods which will improve subgenus identification of adenoviruses.

Mapping of linear epitopes on fibre knob of human adenovirus serotype 5.

Linear antigenic epitopes on the Ad5 fibre knob (FK5) were characterised with fibre- and virion-specific antisera, using 15-mer overlapping peptides covering the knob of the fibre. They were compared with epitopes on the Ad2 fibre knob (FK2) domain. The stronger reactive FK5 epitopes were represented by peptides P3 (amino acids (aa A419-L433)), P6 (aa S449-E463), P7 (aa I459-L473), P12 (aa G509-N523), P14 (aa P529-G543) and P16 (aa A549-Y563). P3 spans the B beta-strand and the left portion of the C beta-strand, P6 and P7 the D beta-strand and the adjacent parts of the CD and DE loops, P12, P14 and P16 the G, H and I beta strands and the adjacent parts of the loops, respectively. The stronger reactive epitopes on FK2 were located in P2 (aa P409-L423), P6 (aa T449-Q463), P8 (aa E469-G483), P13 (aa Q519-T533) and P16 (aa S549-K563). The positions of FK5 and FK2 derived peptides, representing epitopes, are either identical or overlapping or adjacent, as determined by amino acid sequence alignment. Antisera obtained against several longer peptides showed virus neutralising capacity, indicating neutralising epitopes in these peptides.

Adenovirus-receptor interaction with human lymphocytes

Lymphocytes play a key role in cell‐mediated immunity and are host cells for several viral and bacterial pathogens. Their importance in adenovirus (Ad) infections is not yet fully understood. The initial event, the attachment of Ad to lymphocytes and their subsets, was examined using flow cytometry. The study included analysis of stimulated T cells in binding assays with FITC‐labeled Ad fiber. The results confirm that native peripheral lymphocytes express very small amounts of Ad receptors. Stimulation with PHA and interleukin 2 induced the expression. The presence of Ad DNA as a sign of internalization in stimulated cells was demonstrated using the polymerase chain reaction. The findings suggest that lymphocytes after stimulation can turn into target cells for Ad. This is particularly important if there are indications for persistence of Ad, and in the case of immunocompromised patients severe, life‐threatening diseases can develop. J. Med. Virol. 51:252–257, 1997.

Detection of Antibodies against Adenovirus Protein IX, Fiber, and Hexon in Human Sera by Immunoblot Assay

ABSTRACT The 51 serotypes of human adenoviruses (HAdVs) of the genus Mastadenovirus are classified into the six species HAdV-A to HAdV-F. For the detection of genus- and species-specific antibodies in human sera an immunoblot assay was developed. The recombinant long fiber of HAdV-41[F] (Ad41Fi) and the native hexon of HAdV-5[C] were used as genus-specific antigens. The recombinant capsid protein IX (pIX) of HAdV-2 (Ad2pIX[C]) and HAdV-41 (Ad41pIX[F]), the C-terminal pIX part of HAdV-3 (Ad3pIXC[B]), and the fiber knob of HAdV-8 (Ad8FiKn[D]) were evaluated as representative species-specific antigens. Hence, the pIX amino acid sequences of numerous serotypes of all HAdV species were compared, and the cross-reactivities of pIX antigens with rabbit hyperimmune sera among HAdV-A to -F were analyzed. In an epidemiological study, 667 human patient sera, not selected for viral infection, were screened for adenovirus seroprevalence. The genus-specific antibody prevalences directed against the Ad41Fi and HAdV-5 hexon were 82.8 and 98.8%, respectively. The species-specific antibody prevalence of 44.7% against Ad2pIX[C], 36.6% against Ad41pIX[F], 26.4% against Ad8FiKn[D], and 18% against Ad3pIXC[B] showed an age-dependent distribution and correlated well with the frequency of isolated serotypes of the respective species in earlier studies (except HAdV-D). In conclusion, the immunoblot assay using pIX, fiber, and hexon antigens represents a valuable and new serological tool for refined adenovirus diagnosis as shown in an epidemiological study.

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.

Abstract 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.

Mapping of Epitopes on the Fiber Knobs of Human Adenovirus Serotypes 8 and 15

In order to obtain information on the linear antigenic epitopes on fiber knobs of adenovirus serotypes 8 (Ad8) and 15 (Ad15) of subgenus D, the binding of polyclonal virus- and fiber-specific rabbit antibodies to overlapping peptides covering the fiber knob was studied. The main antigenic epitopes of the fiber knob of Ad8 (FK8) were represented by the peptides P4 [amino acids (aa) 213–227], P6 (aa 233–247), P11 (aa 283–297), P13 (aa 303–317) and P15 (aa 316–325); the peptides P1 (aa 183–197), P8 (aa 253–267), P10 (aa 273–287) and P23 (aa 340–349) were moderately reactive. The peptides P4, P6, P11, P13 and P15 span the β strands C, D, G and H, parts of the CD and DG loops and the complete GH loop of the fiber knob. The main epitopes of the fiber knob of Ad15 (FK15) were represented by peptides P5 (aa 198–212), P10 (aa 223–237), P12 (aa 233–247), P13 (aa 238–252), P26 (aa 303–317), P29 (aa 318–332) and P32 (aa 333–347), spanning the β strands B, D, G, H and I, partly strand C, the CD loop, parts of the AB and DG loops, the GH and HI loops and the N-terminal part of the IJ loop. The amino acid sequence alignment showed that the location of the linear FK8 and FK15 epitopes was found to be overlapping to a major extent. Two serotype-specific epitopes were determined on FK15, represented by P10 and P13.

Human CAR Gene Expression in Nonpermissive Hamster Cells Boosts Entry of Type 12 Adenovirions and Nuclear Import of Viral DNA

ABSTRACT Adenovirus type 12 (Ad12) propagation in hamster BHK21 cells is blocked prior to viral DNA replication. The amounts of Ad12 DNA in the nuclei or cytoplasm of hamster cells are about 2 orders of magnitude (2 h postinfection [p.i.]) and 4 to 5 orders of magnitude (48 h p.i.) lower than in permissive human cells. Cell line BHK21-hCAR is transgenic for and expresses the human coxsackie- and adenovirus receptor (hCAR) gene. Nuclear uptake of Ad12 DNA in BHK21-hCAR cells is markedly increased compared to that in naïve BHK21 cells. Ad12 elicits a cytopathic effect in BHK21-hCAR cells but not in BHK21 cells. Quantitative PCR or [3H]thymidine labeling followed by zone velocity sedimentation fails to detect Ad12 DNA replication in BHK21 or BHK21-hCAR cells. Newly assembled Ad12 virions cannot be detected. Thus, the block in Ad12 DNA replication in hamster cells is not released by enhanced nuclear import of Ad12 DNA.