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Background: Around one million people are affected by adenoviral keratoconjunctivitis a year in Japan, and it is recognized as one of the major pathogens of ophthalmological nosocomial infection worldwide. Although cidofovir can be used systemically for immunocompromised patients with disseminated adenoviral infection, no specific anti-adenoviral agent has been established for the treatment of adenoviral infection. We evaluated the anti-adenoviral effect of anti-HIV (human immunodeficiency virus) agents in this study., Methods: Five anti-HIV agents (zalcitabine, stavudine, nevirapine, indinavir and amprenavir) were subjected to in vitro evaluation. A549 cells were used for viral cell culture, and adenovirus serotypes 3, 4, 8, 19 and 37 were used. After calculating CC(50) (50% cytotoxic concentration) of each agent by MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) method, we cultured adenovirus with the agents for seven days and quantitatively measured extracted adenoviral DNA by real-time PCR., Results: Among the anti-HIV drugs, zalcitabine and stavudine, both nucleoside reverse transcriptase inhibitors, showed significant anti-adenoviral activity. In contrast, nevirapine, a non-nucleoside reverse transcriptase inhibitor, and indinavir and amprenavir, which are both protease inhibitors, were ineffective against adenovirus., Conclusions: These results indicate that zalcitabine and stavudine are possible candidates for the local and systemic treatment of adenoviral infection, and the anti-adenoviral effect might depend on the pharmacological properties of anti-HIV agents. The chemical properties on the clinical safety for systemic and local application need to be determined in order to for these drugs to be accepted for the treatment of adenovirus in clinical settings.

Adenovirus (Ad) infection results in significant morbidity and mortality in both immunocompetent and immunosuppressed hosts. There is currently no licensed chemotherapy effective in dealing with this virus infection. In this study the anti-adenoviral activity of a group of modified nucleoside analogs was investigated. The most efficient 3-fluorosubstituted nucleoside triphosphate inhibitors of Ad DNA polymerase were 3'-fluorothymidine triphosphate (IC50 0.63 microM), 2',3'-dideoxy-3'-fluoroguanosine triphosphate (IC50 0.71 microM) and 2',3'-dideoxy-3'-fluorouridine triphosphate (IC50 2.96 microM). The most efficient 2',3'-dideoxynucleoside triphosphates were 2',3'-dideoxycytidine triphosphate (ddCTP; IC50 1.0 microM), 2',3'-dideoxyadenosine triphosphate (IC50 1.6 microM) and 2',3'-dideoxythymidine triphosphate (IC50 1.82 microM). Kinetic studies indicate competitive inhibition of adenovirus DNA polymerase by ddCTP. These data confirm results previously obtained at the cellular level using a focus reduction assay involving Ad2-infected FL cells. Whereas the D-enantiomers 3'-fluorothymidine and 2',3'-dideoxycytidine are potent inhibitors of adenoviral replication, the corresponding L-enantiomers exhibited no inhibitory activity.

HeLa cells were depleted of 75% of the available adenovirus type 2 specific cellular receptor sites (CRSs) by controlled attachment of viruses at a high multiplicity of infection. Upon removal of unattached viruses and further incubation, the cells were able to recycle the CRSs to 75% of the level of uninfected control cells within 5 h. The rate of virus receptor recycling was approx. 1 CRS x min-1 x cell-1. The existence of receptor recycling further strengthens the involvement of a total process of receptor-mediated endocytosis in adenovirus internalization. The recirculation process was neither affected by the lysosomotropic agents ammonium chloride and amantadine-HCl, nor by the ionophore monensin or the multifunctional weak-base amine chloroquine.

Periodate oxidation of purified type 5 Adenovirus (Ad5) led to a mean loss of infectivity of 6.84 logs. There were no significant differences in adsorption and penetration between oxidized and mock-oxidized virus. However, after infection with oxidized virus, no synthesis of viral structural proteins could be detected and a 78.5% inhibition of viral DNA synthesis was observed. Labelling experiments performed by treating oxidized and mock-oxidized virus with tritiated sodium borohydride revealed that the fiber glycoprotein was one of the proteins labelled in oxidized virus whereas no labelled proteins were detected in non oxidized virus. In addition, it was found that one mol of formaldehyde generated during oxidation of sugar residues was bound per 500 base pairs in oxidized virus. One consequence of this in situ generation of formaldehyde is the formation of DNA-protein crosslinks. The DNA so crosslinked showed different patterns of restriction fragments with endonucleases such as Hpa I, Hind III and Kpn I but not with Xho I.

Estramustine phosphate, an estradiol-mustard conjugate, was shown to reversibly inhibit a stage during the first hour of productive adenovirus 2 infection of HeLa cells. This drug, employed in the therapy of advanced prostatic cancer, specifically interacts with microtubule-associated proteins (MAPs) of the cytoskeleton. The results obtained under physiological conditions in vivo suggest a MAPs-interference with the microtubule-mediated vectorial migration of the virus inoculum to the nucleus. Virus attachment, uncoating kinetics and the appearance of established uncoating intermediates were not affected.

"Freeze-fracture negative staining" of adenovirus type 5 revealed the virus cores as internal bodies with a fine granular surface which at high magnification shows reticular and ring-like patterns. This indicates that the virus protein V forms a thin surface layer for which a name "core shell" is proposed. Virus cores prepared by heating virus particles in sodium deoxycholate (DOC) relaxed into curved filaments or several rods by means of EGTA and high salt, respectively. High pH treatment had similar effects as high salt. Rod-like elements were also observed in ultrathin sections of the "DOC-cores". Fresh cores exhibited circular dichroism (C.D.) with similar features as described for nucleosomes (COWMAN and FASMAN, ref. 5). This indicates that the DNA has an orderly arrangement in the cores. EGTA had no effect on C.D. spectra but high salt treatment abolished the positive peak in 10 minutes. It is concluded that the adenovirus nucleocapsid is a linear structure, presumably a 120-150 Angstrom thick filament folded 5 to 6 times into "rods" as previously observed by freeze-fracturing.

The antiviral action of a new drug, 5-amino-2-(dimethylaminoethyl)-benzo-[de]-isoquinolin-1.3-dione has been studied against herpes simplex type 2 (HSV-2) and adenovirus type 5 (Ad-5) grown in Vero cells. The concentration of the drug which gives a 5 log10 reduction in virus titer was 4 micrograms/ml (maximum tolerated concentration) for HSV-2. The anti-HSV-2 activity of this compound was one log. more powerful than that of iododeoxyuridine (IDU). At this concentration the drug shows virucidal activity against HSV-2. No inhibition was found when the drug was tested against Ad-5. The inhibition of virus production has been studied depending upon the amount of drug or virus, and drug addition-time after infection. Reversibility of inhibition after drug removal, and drug resistance in the presence of the drug have also been determined.