Recombinant adenoviruses are presently the most efficient in vivo gene transfer system available. Targeting single organs or large tumors by adenoviral vectors requires an intravascular route of application. During the first pass of viral particles through the vascular bed of the target tissue, virus uptake is not quantitative and undefinite amounts of particles leak into circulation. To determine the amount of leaking particles and to calculate organ-specific uptake (in-/outflow ratio), it is necessary to titrate virus particles directly in blood. In preclinical and clinical trials titration is currently mostly done with blood plasma instead of full blood. However, this technique provides valid results only as long as there is no affinity between adenovirus particles and erythrocytes. In this study we demonstrate that Ad5 particles, as mostly employed for gene therapy, have a strong affinity to human erythrocytes. At 60 min after coincubation of human erythrocytes and Ad5 particles, more than 98% of the particles are attached to the surface of erythrocytes. Therefore, ignoring the amount of red cell bound particles by performing titration in plasma leads to severe miscalculation of organ-specific transfer rates or virus circulation half-life. The biological impact of an increased affinity between virus particles and erythrocytes will be discussed.

Rapid diagnosis of human adenovirus (HAdV) infections was achieved by PCR in the recent years. However, conventional PCR has the risk of carry‐over contamination due to open handling with its products, and results are only qualitative. Therefore, a quantitative “real‐time” PCR with consensus primer and probe (dual fluorescence labelled, “TaqMan”) sequences for a conserved region of the hexon gene was designed and evaluated. Real‐time PCR detected all 51 HAdV prototypes. Sensitivity of the assay was ≤15 copies/run and the linear range of quantitation 1.5 × 101 to 1.5 × 108 copies/run. TaqMan PCR gave identical results compared to an established conventional one‐step PCR protocol in 218 (38 positive and 180 negative) of 234 clinical samples including blood, serum, eye swabs, and feces, and had divergent results in 16 samples (15 positive only in TaqMan PCR, all with low copy numbers, and one positive only in conventional PCR), indicating a higher sensitivity of TaqMan PCR. Adenovirus viremia was detected by TaqMan PCR in 4 of 27 (14.8%) paediatric and 8 of 93 (8.6%) adult stem cell transplant recipients but only in 5 of 306 healthy controls (blood donors, 1.6%). Virus loads of pediatric patients (median 1.7 × 105) were significantly higher than in adult patients (median 2.3 × 103) and than in controls (all samples ≤1.7 × 103 copies/ml). A few immunosuppressed children had very high virus loads (up to 1.1 × 1010 copies/ml), which were associated with symptoms of disseminated disease. In conclusion, real‐time PCR is a sensitive and quantitative procedure for the detection of adenovirus infections. J. Med. Virol. 70: 228–239, 2003. © 2003 Wiley‐Liss, Inc.

ABSTRACT Epidemic keratoconjunctivitis (EKC) is a severe eye infection caused mainly by adenovirus type 8 (Ad8), Ad19, and Ad37. We have shown that the EKC-causing adenoviruses use sialic acid as a cellular receptor on A549 cells instead of the coxsackie-adenovirus receptor, which is used by most adenoviruses. Recently, Wu et al. (Virology 279:78-89, 2001) proposed that Ad37 uses a 50-kDa protein as a receptor on Chang C conjunctival cells and that this interaction is independent of sialic acid. According to the American Type Culture Collection, this cell line carries HeLa cell markers and should be considered to be a genital cell line. This prompted us to investigate the function of sialic acid as a cellular receptor for Ad37 in Chang C cells. In this study, we demonstrate that enzymatic removal or lectin-mediated blocking of cell surface sialic acid inhibits the binding of Ad37 virions to Chang C cells, as does soluble, virion-interacting sialic acid-containing substances. The binding was Ca 2+ or Mg 2+ ion independent and mediated by the knob domain of the trimeric viral fiber polypeptide. Moreover, Ad37 virions infected Chang C cells and two other genital cell lines (HeLa and SiHa) as well as a corneal cell line in a strictly sialic acid-dependent manner. From these results, we conclude that Ad37 uses sialic acid as a major receptor in cell lines derived from both genital and corneal tissues.

Recombinant adenoviruses are currently the most important vector system in gene therapy. Adenoviruses frequently cause upper respiratory tract infections in humans and anti-adenoviral antibodies are found in 35–70% of the population. Therefore in the majority of potential patients receiving adenoviral gene therapy, the contact of virus particles and blood will lead to the formation of antigen–antibody complexes. These complexes have the ability to induce inflammatory reactions via an activation of the complement system. We have determined the level of C3a (the most reactive complement component) generated in isolated citrate plasma of healthy individuals after challenge with recombinant and wild-type adenoviruses in amounts corresponding to virus blood levels to be expected in patients during adenoviral gene therapy. All plasma samples containing anti-adenoviral antibodies showed a substantial, dose-dependent generation of C3a. A virus plasma level of about 7.5 × 109 particles/ml (which was calculated to be the highest blood level reached during clinical trials in the past) induced an average release of about 3000 ng/ml C3a (baseline levels <140 ng/ml). Analyzing the nature of anti-adenoviral antibodies showed, that not only antibodies with neutralizing properties (anti-Ad5), but also non-neutralizing anti-adenoviral antibodies are capable of complement activation. This study suggests that complement activation can be ignored in local low-dose applications of recombinant adenoviruses, but warrants attention after systemic application of large viral quantities. In clinical protocols aiming at systemic virus application, measures for monitoring and controlling the complement system should be included on a regular basis.

The polymerase chain reaction (PCR) has been used previously for the detection acid typing of adenoviruses directly in clinical samples. Since under clinical conditions subgenus-specific identification is often sufficient, we extended the genus- and type-specific PCR by a subgenus-specific PCR. By sequencing several loop I, gene regions of the hexon (major adenovirus coat protein) and comparing them to published sequences, subgenus-specific sequences were identified in this region. By using primers targeted to this region and to a conserved hexon gene region, a multiplex, nonnested PCR for the detection and subgenus-specific identification of adenoviruses could be established. The six subgenus-specific amplimers are distinguishable by agarose gel electrophoresis, and subsequent restriction analysis is not necessary. The specificity of the subgenus-specific primer pairs was tested on 23 adenovirus prototypes, representing all six subgenera, on 9 subgenus B and D intermediate strains, and on 16 subgenus C genome types. Furthermore, multiplex, subgenus-specific PCR was performed directly with 100 clinical specimens, including stool samples, ocular swabs, and throat swabs. Adenoviruses of all subgenera could be detected. Especially for clinical application, the rapid, one-step differentiation between subgenus D adenoviruses, causing the severe and highly contagious epidemic keratoconjunctivitis, and subgenus B and E adenoviruses, causing relative harmless ocular infections, is of great importance. The subgenus-specific PCR could also facilitate the primary classification of unknown virus isolates. J. Med Virol. 58:87-92, 1999. (C) 1999 Wiley-Liss, Inc.

BACKGROUND:Enteroviruses were detected in up to 50% in myocardium of patients with myocarditis and dilated cardiomyopathy, the latter being considered as a result of a prior subclinical myocarditis. A wide range of other infectious agents are being discussed as pathogens, often only based on reports of single cases. Adenovirus genome was recently identified in a significant number in the myocardium of paediatric patients with myocarditis. However, data on the role of adenoviruses for the aetiopathogenesis of myocarditis in adult patients is missing so far. Therefore, we studied the prevalence of adenoviral and enteroviral genome in myocardium of adults with myocarditis and dilated cardiomyopathy.METHODS:15 patients were diagnosed at baseline with myocarditis, 16 patients with dilated cardiomyopathy according to clinical and histological criteria. Endomyocardial biopsies of these patients and 8 control patients with non-infectious heart diseases were evaluated by polymerase chain reactions for enterovirus and adenovirus genome.RESULTS:Enteroviral genome was detected in 27.3% patients with myocarditis or dilated cardiomyopathy, whereas adenoviral genome was not identified in any patient. Samples from control subjects systematically yielded negative results.CONCLUSIONS:From our data, it seems doubtful that adenoviruses are major pathogens of myocarditis or DCM, whereas enterovirus genome was identified in a significant number of patients with both diseases.

Poliovirus type 1 was isolated from an immunodeficient patient 4 days after onset of paresis (IS1) and after 5.5 years of prolonged enteral virus replication (IS2). Antigenic characterization revealed that IS1 was Sabin 1-like, whereas IS2 reacted like poliovirus 1 Mahoney. Complete genomic sequencing demonstrated the phylogenetic relationship (94.9% identity) of IS1 and IS2, which differed from the most closely related Sabin 1 by 5.4 and 8.3%, respectively. Both isolates had revertant-like mutations at nucleotides 480 and 6203. Deduced amino acid sequences indicated significant changes between IS1 and IS2 at the neutralizing antigenic site 1. Prolonged enteral replication, evolution, and shedding of poliovirus by immunodeficient patients should be considered in the poliovirus eradication strategy.

ABSTRACT The adenovirus fiber knob causes the first step in the interaction of adenovirus with cell membrane receptors. To obtain information on the receptor binding site(s), the interaction of labeled cell membrane proteins to synthetic peptides covering the adenovirus type 3 (Ad3) fiber knob was studied. Peptide P6 (amino acids [aa] 187 to 200), to a lesser extent P14 (aa 281 to 294), and probably P11 (aa 244 to 256) interacted specifically with cell membrane proteins, indicating that these peptides present cell receptor binding sites. Peptides P6, P11, and P14 span the D, G, and I β-strands of the R-sheet, respectively. The other reactive peptides, P2 (aa 142 to 156), P3 (aa 153 to 167), and P16 (aa 300 to 319), probably do not present real receptor binding sites. The binding to these six peptides was inhibited by Ad3 virion and was independent of divalent cations. We have also screened the antigenic epitopes on the knob with recombinant Ad3 fiber, recombinant Ad3 fiber knob, and Ad3 virion-specific antisera by enzyme-linked immunosorbent assay. The main antigenic epitopes were presented by P3, P6, P12 (aa 254 to 269), P14, and especially the C-terminal P16. Peptides P14 and P16 of the Ad3 fiber knob were able to inhibit Ad3 infection of cells.

The adenovirus fiber mediates the agglutination of erythrocytes. Based on differential hemagglutinating properties, subgenus D adenoviruses can be subdivided into clusters DI, DII, and DIII. While subgenus DI adenoviruses agglutinate rat and human erythrocytes, DII adenoviruses simply agglutinate rat erythrocytes and DIII adenoviruses display no or only weak rat erythrocyte agglutination. Amino acid sequence comparisons revealed distinct domains on the fiber knob which could be involved in hemagglutination. In order to localize and characterize the domains responsible for the interaction with rat and human erythrocytes, potential hemagglutination domains of the adenovirus type 9 (Ad9) (subgenus DI) fiber knob were introduced into Ad17 (subgenus DII) and Ad28 (subgenus DIII) fiber knobs by primer-directed mutagenesis. Furthermore, rat erythrocyte hemagglutination domains were also introduced into the Ad3 (subgenus B) fiber knob, which only agglutinated monkey erythrocytes. Altogether, 27 chimeric and mutated fiber proteins were expressed in Escherichia coli and subsequently tested for hemagglutination activity. The hemagglutination tests revealed that at least two domains can mediate the agglutination of rat erythrocytes. While one domain is located on the GH loop, the other domain extends from the C beta strand to the CD loop. The domain on the GH loop was partially conserved in all adenoviruses showing an incomplete hemagglutination pattern with rat erythrocytes. The domains involved in the agglutination of human erythrocytes are located on the CD and HI loops of the subgenus DI fiber knob.

Since human adenoviruses (Ad) are associated with a variety of diseases, there is need for a fast and sensitive diagnostic procedure. The polymerase chain reaction (PCR) has been previously applied for the detection and typing of adenoviruses directly in clinical samples, So far, only Ad8, Ad31, Ad40 and Ad41 could be typed by PCR. To extend the technique of type-specific PCR to other adenovirus serotypes, type-specific primers for Ad1, Ad2, Ad4, Ad5, Ad19 and Ad37 were evaluated. In the present study, 50 stool and 68 eye swab specimens were first tested for the presence of adenoviruses using genus-specific primers. Adenoviruses could be detected in 42 stool and 47 eye swab samples. While the adenovirus-positive stool samples were subsequently typed with primers for Ad2, Ad5, Ad31, Ad40 and Ad41, the positive eye swab specimens were typed with primers for Ad4, Ad8, Ad19 and Ad37.

As enterovirus infections of the heart cause myocarditis and eventually congestive heart failure, the antiviral activity of ribavirin was studied in coxsackie virus B3 (CVB3)-infected carrier cultures of human myocardial fibroblasts. Cultures were infected 7 days before application of ribavirin and effects were evaluated over a period of 16 days by plaque assays and in situ hybridization. Compared to the low antiviral activity in HeLa cells, ribavirin was highly active in reducing infectious virus yields in human myocardial fibroblasts, for example, to 2.0 x 10(3) pfu/ml with 25 microg/ml and to 1.3 x 10(2) pfu/ml with 50 microg/ml (4.3 x 10(4) pfu/ml in infected controls). Moreover, 100 microg ribavirin/ml completely suppressed infectious virus progeny in two of three cultures, and reduced the number of infected cells from 14.3 to 0.3% as determined by in situ hybridization, whereas up to 3200 microg ribavirin/ml did not result in a significant cytotoxic effect. Interaction with interferon-alpha (IFN-alpha) was additive to slightly synergistic in reducing the number of infected cells and virus yields. In conclusion, our results suggest a cell-specific high activity of ribavirin in human myocardial fibroblasts and indicate the importance of using organ-specific cells for testing antiviral agents in myocarditis. Furthermore, the usefulness of in situ hybridization for determining the long term effects of antivirals in carrier state cell cultures was demonstrated.

Lack of in vitro cultivation methods has inhibited the development of rapid, reliable diagnostic procedures for adenovirus-associated necrotizing bronchopneumonia in guinea pigs. Because polymerase chain reaction (PCR) techniques are well established for human adenoviruses, primers for the amplification of guinea pig adenovirus DNA were evaluated. The DNA for PCR was purified from the lung tissue of spontaneously infected and healthy guinea pigs. Adenovirus DNA could only be detected in the lungs of the infected animals. Subsequent sequence analysis of PCR products revealed that the guinea pig adenovirus is a distinct adenovirus.

The fiber knob carries the type-specific gamma-antigen which can be demonstrated in hemagglutination inhibition tests. In order to characterize the gamma-determinant we selected subgenus DI adenovirus serotypes 9 and 19 (Ad9 and Ad19) which exhibited 29 amino acid exchanges in the knob domain. Like all subgenus DI adenoviruses they showed a complete hemagglutination pattern with rat and human erythrocytes. We constructed a total of 14 chimeric Ad9/Ad19 and Ad19/Ad9 fiber proteins, which possessed fiber knobs with progressively exchanged Ad9 and Ad19 amino acids. Furthermore, we created 39 fiber proteins with distinct amino acid exchanges in the knob regions by primer-directed mutagenesis. The proteins were expressed in Escherichia coli and tested in hemagglutination and hemagglutination inhibition tests. From our results we can conclude that the type-specific gamma-determinant is not restricted to a distinct region on the adenovirus fiber knob but is composed of at least 17 amino acids. Most of the amino acids contributing to the Ad9 and Ad19 gamma-determinants are located on the fiber knob loops.

The adenovirus fibre carries the type-specific gamma determinant the existence of which was suggested by haemagglutination inhibition tests. Furthermore, the fibre is thought to be responsible for the agglutination of monkey, rat and human erythrocytes. In order to verify that the haemagglutination properties and the type-specific gamma determinant are located on the fibre knob domain, several recombinant fibre proteins of subgenus D adenoviruses were constructed, expressed in HeLa cells and tested in haemagglutination and haemagglutination inhibition tests. Our data showed that the epitopes responsible for the interaction with erythrocytes and the gamma determinant are located on the fibre knob domain.

The fiber gene sequences of the human adenovirus types 19 and 37 were determined. The predicted polypeptides exposed a high homology. Ad8, Ad19, and Ad37 can cause epidemic keratoconjunctivitis (EKC), whereas Ad9 only infrequently causes acute follicular conjunctivitis. Comparison of the fiber knobs of Ad8, Ad9, Ad19, Ad37, and other previously published fiber sequences revealed that only two amino acid residues were conserved in the fiber knobs of Ad8, Ad19, and Ad37. Since the knob is responsible for interaction with the cell receptor, these two amino acid residues could play an important role in the pathogenicity of EKC causing adenoviruses.

Detection of enterovirus RNA in endomyocardial biopsies (EMB) by reverse transcription/polymerase chain reaction (RT-PCR) is currently the preferred diagnostic procedure in suspected enterovirus heart disease (EHD), which can present clinically as myocarditis, dilated cardiomyopathy (DCM), and arrhythmogenic right ventricular cardiomyopathy (ARVC). EMB and peripheral blood mononuclear cells (PBMC) of 44 patients with suspected EHD were examined by nested RT-PCR to investigate whether the myocardial enterovirus infection is limited to the heart or is generalized. Enterovirus RNA was detected in EMB, but not in PBMC, of 8 patients (3 of these suffered from ARVC), whereas EMB of 16 controls and PBMC of 45 controls were negative. In addition, enterovirus RNA was demonstrated in PBMC, but not in EMB, of a single patient with suspected EHD. A high sequence homology of the amplicons to coxsackievirus B3 was demonstrated in 7 patients, and to coxsackievirus B2 in two patients. In order to evaluate whether the myocardial enterovirus infection was acute or persistent, neutralization and complement fixation tests were performed for antibodies against the serotypes indicated by the nucleic acid sequences. Neutralizing antibodies were detected in the sera of all 9 patients, but complement fixing antibodies were demonstrated only in one EHD patient and in the patient positive for enterovirus RNA in PBMC. In conclusion, the molecular and serological data demonstrate that CVB3 predominates as cardiotropic enterovirus, and that the enterovirus replication is limited to the heart in EHD. Serological results support the hypothesis of myocardial enterovirus RNA persistence in spite of neutralizing antibodies.

We compared the antiviral activities of three recombinant human interferons (IFN-alph2a, IFN-beta, and IFN-gamma) in cultured human myocardial fibroblasts to select a candidate for trial in heart disease induced by cardiotropic enterovirus, e.g., coxsackievirus B3 (CVB3). Cells were exposed to CVB3, and after 7 days, when a persistent infection had developed, IFN was added. Virus yields were measured on alternate days for the next 7 or 16 days, and IFN activity was assessed as the percentage reduction in yield. IFN-gamma and IFN-beta were both highly active and reduced virus yields by 2 log (EC(99)) at concentrations of 23.4 IU/ml (SD = 8.6) and 10.1 IU/ml (SD = 3.2), respectively; with 250 IU/ml of either IFN, no infectious virus was formed. Unexpectedly, IFN-alpha2a (EC(99)> 1250 IU/ml) was at least 120 times less active than IFN-beta; after use for 8 days or more, the minor effects it produced were no longer related to the concentration applied. Despite the pharmacokinetic advantages of IFN-alpha2a, our data suggest that IFN-beta should in preference be evaluated in the clinic.

Respiratory tract infections have been thought to act as a trigger mechanism in sudden infant death. In 118 autopsy cases of infant death, paraffin-embedded or frozen lung tissues were investigated by means of a nested polymerase chain reaction (PCR) to detect adenovirus (AV) DNA. The primers used are general primers and allow the detection of most pathogenic adenoviruses with high specificity and sensitivity and independently of devitalization of viruses or degradation of viral DNA. For the investigation three groups were established: there were 13 cases of unnatural death, 78 cases of natural death without histological signs of interstitial pneumonia, and 27 cases with interstitial pneumonia. The first group was AV negative. In the group without interstitial pneumonia AV was detected in 10.2% of the cases. In the group with interstitial pneumonia the frequency of AV detection was almost 26%. The results obtained demonstrate an association between interstitial pneumonia and detection of AV DNA, indicating that AV may play an important part in pulmonary infection in infants. Histological evidence of interstitial pneumonia was not observed in all AV-positive cases, perhaps because nonspecific virus-related changes occurred only in early stages of viral infection. Comparison of the AV frequency in SIDS (15%) and non-SIDS cases (4%) indicates an association between pulmonary AV infections and sudden death. These results support the working hypothesis of respiratory infections acting as a trigger mechanism in sudden infant death.