Coxsackievirus A24 variant uses sialic acid-containing O-linked glycoconjugates as cellular receptors on human ocular cells.

Coxsackievirus A24 variant (CVA24v) is a main causative agent of acute hemorrhagic conjunctivitis (AHC), which is a highly contagious eye infection. Previously it has been suggested that CVA24v uses sialic acid-containing glycoconjugates as attachment receptors on corneal cells, but the nature of these receptors is poorly described. Here, we set out to characterize and identify the cellular components serving as receptors for CVA24v. Binding and infection experiments using corneal cells treated with deglycosylating enzymes or metabolic inhibitors of de novo glycosylation suggested that the receptor(s) used by CVA24v are constituted by sialylated O-linked glycans that are linked to one or more cell surface proteins but not to lipids. CVA24v bound better to mouse L929 cells overexpressing human P-selectin glycoprotein ligand-1 (PSGL-1) than to mock-transfected cells, suggesting that PSGL-1 is a candidate receptor for CVA24v. Finally, binding competition experiments using a library of mono- and oligosaccharides mimicking known PSGL-1 glycans suggested that CVA24v binds to Neu5Acα2,3Gal disaccharides (Neu5Ac is N-acetylneuraminic acid). These results provide further insights into the early steps of the CVA24v life cycle.

Anti-adenoviral effect of interferon-ß and interferon-γ in serotypes that cause acute keratoconjunctivitis.

BACKGROUND: Interferons are currently used for hepatitis B and C virus infection; the effect of interferons against adenovirus has not been elucidated. It has been reported that interferon-ß and interferon-γ were effective against adenovirus serotype 3. We investigated the anti-adenoviral effect of interferon-ß and interferon-γ in serotypes that cause acute keratoconjunctivitis, using quantitative polymerase chain reaction in vitro. DESIGN: Experimental study carried out in a university. SAMPLES: Five strains of different serotypes of adenovirus, types 3 (AdV3; species B), 4 (species E), 8, 19a and 37 (species D) and interferon-ß and interferon-γ for in vitro assay. METHODS: After pretreatment of A549 with serial dilution of interferons for 24 h, adenovirus was cultivated for 7 days, and adenoviral DNA was quantitatively measured by real-time polymerase chain reaction. MAIN OUTCOME MEASURES: Number of virus copies after treatment and the 50% effective concentration (EC(50) ). RESULTS: Interferon-ß showed a dose-dependent inhibitory effect on all serotypes. EC(50) of interferon-ß ranged between 211 and 843 IU/mL. A similar tendency was observed with interferon-γ. EC(50) of interferon-γ ranged between 133 and 9130 IU/mL. Among the serotypes, interferon-γ exhibited the greatest inhibitory effect on AdV37. In contrast, AdV4 showed the lowest sensitivity to interferon-γ. Statistically significant dose dependency for both interferon-ß and interferon-γ was observed in several serotypes. CONCLUSIONS: These results suggest that both interferon-ß and interferon-γ have anti-adenoviral activity in vitro. Interferons have the potential to be used for local treatment of adenoviral keratoconjunctivitis, although further evaluation in animal models and clinical trials is necessary.

Clinical evaluation of local ocular toxicity in candidate anti-adenoviral agents in vivo.

BACKGROUND: Adenovirus causes ophthalmological nosocomial infections. Although cidofovir may be used systemically for immunocompromised patients in disseminated adenoviral infections, no specific anti-adenoviral agent has been established for the treatment of adenoviral ocular infection. It has been reported that cidofovir may cause lacrimal duct obstruction when used locally. We have reported that zalcitabine and stavudine showed anti-adenoviral activity in vitro. We now evaluate the side effects of these agents in eyes and ocular adnexa in an animal model. METHODS: Cidofovir, zalcitabine and stavudine 1% solutions and balanced salt solution were given as eye drops to healthy female Japanese albino rabbits 4 times a day for 14 days. Clinical scores in the conjunctiva and eyelid were recorded, and lacrimal irrigation was carried out. The diameter of the lacrimal canaliculus was scanned with an ultrasonograph. Histopathological analysis was carried out in the cidofovir group. RESULTS: In the cidofovir group, significant narrowing of lacrimal canaliculus, redness of eyelids and conjunctival injection were observed, but no obstruction of the lacrimal duct was found. Histology showed eosinophils, suggesting an allergic inflammation. Although zalcitabine and stavudine induced eyelid redness and conjunctival injection, no change was observed in the lacrimal pathway. CONCLUSIONS: These drugs may be possible candidates as eye drops for adenoviral conjunctivitis but further study is required to prove its safety.

[Long-term time series analysis of epidemic keratoconjunctivitis in Okinawa Prefecture, Japan].

PURPOSE: Epidemic keratoconjunctivitis (EKC) is a highly infectious disease caused by an adenovirus and is associated with nosocomial or endemic infections. In a series analysis before 1993, we predicted that EKC patients would decrease in Okinawa. We analyzed the number of EKC patients after 1993 in the same area to confirm the compatibility between measured and predicted epidemic curves. SUBJECTS AND METHODS: The numbers of EKC patients reported by the National Epidemic Surveillance Report of Japan between 1993 and 2003 in Okinawa were analyzed by time series analysis. RESULTS: The epidemic curve showed a peak in late 1995 and the number decreased thereafter. Among epidemic tendency curves, an exponential curve fitted best to the measured curve and this result was compatible with our past prediction. CONCLUSION: The similarity of the epidemic curve analyzed by long-term time series analysis and the previously predicted curve of EKC in Okinawa, Japan, supports the epidemiological hypothesis that Okinawa is an epidemiologically isolated island for EKC, and its population can be deemed a theoretical epidemiologically closed group.

Anti-adenoviral effects of human cationic antimicrobial protein-18/LL-37, an antimicrobial peptide, by quantitative polymerase chain reaction.

PURPOSE: Antimicrobial peptides have an important role in self-protection of the ocular surface. Human cationic antimicrobial protein (hCAP)-18 is a linear, α-helical peptide that consists of a conserved pro-sequence called a cathelin-like domain and a C-terminal peptide named LL-37. We investigated the in vitro anti-adenoviral activity of hCAP-18/LL-37 in several adenovirus types, inducing keratoconjunctivitis. METHODS: A549 cells were used for viral cell culture, and human adenovirus (HAdV) types 3 (HAdV3, species B), 4 (species E), 8, 19a, and 37 (species D) were used. The cytotoxicity of LL-37 was evaluated by 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) assay to obtain 50% cytotoxic concentration. After pretreatment of A549 cells with serial dilutions of LL-37 for 24 hours, adenovirus was cultured for seven days, and adenoviral DNA was quantitatively measured by real-time polymerase chain reaction (PCR). RESULTS: The 50% effective concentration of LL-37 obtained by real-time PCR ranged between 118 and 270 µM. LL-37 showed a significant inhibitory effect on adenoviral proliferation in all adenovirus types except HAdV4 in a dose-dependent manner. CONCLUSIONS: LL-37 has significant inhibitory activity against HAdV3, 8, and 19, which induce keratoconjunctivitis. These results indicate that hCAP-18/LL-37 may be a possible candidate for the treatment of HAdV keratoconjunctivitis.

Antiadenoviral effects of N-chlorotaurine in vitro confirmed by quantitative polymerase chain reaction methods.

PURPOSE: Adenoviral keratoconjunctivitis is recognized as one of the major pathogens of ophthalmological nosocomial infection worldwide. N-Chlorotaurine (Cl-HN-CH(2)-CH(2)-SO(3)H, NCT) is the N-chloro derivative of the amino acid taurine, which is an oxidant produced by human granulocytes and monocytes during inflammatory reactions. Using conventional viral plaque assay, it was previously shown that NCT causes inactivation of several human adenovirus (HAdV) serotypes. In this study, we evaluated the antiadenoviral effect of NCT by quantitative polymerase chain reaction (PCR) methods. METHODS: A549 cells were used for viral cell culture, and HAdV serotypes 3, 4, 8, 19, and 37 were used. After calculating 50% cytotoxic concentration (CC(50)) of NCT by MTS (3-(4,5- dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) method, HAdV was cultured with NCT for 7 days, and extracted adenoviral DNA was quantitatively measured by real-time PCR. RESULTS: A statistically significant (P < 0.05) dose-dependent inhibition was indicated for all serotypes except HAdV type 4 (HAdV4), which was maximally inhibited by only ~50%. Among the serotypes, NCT was particularly effective against HAdV8, HAdV19a, and HAdV37. The 50% effective concentration (EC(50)) obtained by real-time PCR of NCT ranged between 49 and 256 µM. EC(50) of NCT against HAdV3 was slightly higher than that against serotypes of species D. The selective index (CC(50)/EC(50)) ranged between 41 and 60 except for HAdV4 (11.5). CONCLUSIONS: These results show that NCT has an antiviral effect against most serotypes of human HAdV inducing keratoconjunctivitis, indicating its possible therapeutic use.