Enforced Dimerization of CD45 by the Adenovirus E3/49K Protein Inhibits T Cell Receptor Signaling.

Human adenoviruses (HAdVs) are widespread pathogens that generally cause mild infections in immunocompetent individuals but severe or even fatal diseases in immunocompromised patients. In order to counteract the host immune defenses, HAdVs encode various immunomodulatory proteins in the early transcription unit 3 (E3). The E3/49K protein is a highly glycosylated type I transmembrane protein uniquely expressed by species D HAdVs. Its N-terminal ectodomain sec49K is released by metalloprotease-mediated shedding at the cell surface and binds to the receptor-like protein tyrosine phosphatase CD45, a critical regulator of leukocyte activation and functions. It remained elusive which domains of CD45 and E3/49K are involved in the interaction and whether such an interaction can also occur on the cell surface with membrane-anchored full-length E3/49K. Here, we show that the two extracellular domains R1 and R2 of E3/49K bind to the same site in the domain d3 of CD45. This interaction enforces the dimerization of CD45, causing the inhibition of T cell receptor signaling. Intriguingly, the membrane-anchored E3/49K appears to be designed like a "molecular fishing rod" using an extended disordered region of E3/49K as a "fishing line" to bridge the distance between the plasma membrane of infected cells and the CD45 binding site on T cells to effectively position the domains R1 and R2 as baits for CD45 binding. This design strongly suggests that both secreted sec49K as well as membrane-anchored full-length E3/49K have immunomodulatory functions. The forced dimerization of CD45 may be applied as a therapeutic strategy in chronic inflammatory disorders and cancer. IMPORTANCE The battle between viruses and their hosts is an ongoing arms race. Whereas the host tries to detect and eliminate the virus, the latter counteracts such antiviral measures to replicate and spread. Adenoviruses have evolved various mechanisms to evade the human immune response. The E3/49K protein of species D adenoviruses mediates the inhibition of immune cell function via binding to the protein tyrosine phosphatase CD45. Here, we show that E3/49K triggers the dimerization of CD45 and thereby inhibits its phosphatase activity. Intriguingly, the membrane-anchored E3/49K seems to be designed like a "molecular fishing rod" with the two CD45 binding domains of E3/49K as baits positioned at the end of an extended disordered region reminiscent of a fishing line. The adenoviral strategy to inhibit CD45 activity by forced dimerization may be used for therapeutic intervention in autoimmune diseases or to prevent graft rejection after transplantation.

Epidemic keratoconjunctivitis: efficacy of outbreak management.

PURPOSE: Epidemic keratoconjunctivitis (EKC) is one of the most severe ocular viral infections. The aim of this interruptive time series study was to quantitatively evaluate the effectiveness of a hygienic EKC outbreak management concept developed in our ophthalmological department. METHODS: All patients with suspected EKC in the period from August to November 2018 were included in the study. Data were retrospectively collected from the patient's medical documents and records. The disease was diagnosed clinically and confirmed by virus detection through polymerase chain reaction (PCR) from conjunctival swabs. With the beginning of the epidemic, an outbreak management plan was implemented to reduce the nosocomial spread. RESULTS: The outbreak lasted 77 days (20th August 2018 to 4th November 2018) and affected a total of 120 patients. This corresponds to a mean of 1.5 patients per outbreak day. The median age was 58 [1-92] years. Of all patients, 61 (50.8%) were female. Conjunctival swabs were collected in 100/120 (83.3%) cases, the adenovirus being detected in all positive smears (63/63, 100%). The implementation of our outbreak management plan reduced significantly the number of EKC cases per outbreak day and resulted in a reduction of the basic reproduction number by a factor of 2.2. CONCLUSION: The detection of EKC together with the immediate implementation of hygienic outbreak measures can significantly reduce the spread of infection. The implementation of a strict outbreak management concept can significantly reduce the number of EKC cases, thus avoiding possible complications and therefore unnecessary health-related costs.

Multi-parametric evaluation of ocular surface disorders during healing process of viral conjunctivitis.

Purpose To evaluate the ocular surface properties in epidemic keratoconjunctivitis (EKC) patients during healing process, and to detect the damage on conjunctival goblet cells. Methods Bilateral EKC patients confirmed with polymerase chain reaction (PCR) testing were included. Firstly (Group 1) and secondly (Group 2) affected eyes were compared. Ocular surface parameters were performed at the first visit and first month. Results The study included 34 eyes of 17 patients. The mean age was 44.54 ± 16.80 (21-70) years (FM/M 20/14). The ocular findings in Groups 1 and 2 were not significant. For Groups 1 and 2, OSDI was 53.53 ± 23.01 and 35.90 ± 22.19 (p 0.03), tear osmolarity was 309.12 ± 19.38 and 297.47 ± 8.27 mOsm/µL (p 0.029), OSSS was 1.00 ± 0.79 and 0.18 ± 0.39 (p 0.001), T-BUT was 3.59 ± 2.29 and 6.00 ± 1.83 s (p 0.002), and Schirmer's 1 test was 10.94 ± 8.42 and 16.76 ± 9.05 mm (p 0.061), respectively. In Groups 1 and 2, the IC was Grade (G) 0 in 23.5% and 17.6%, G1 in 35.3% and 41.2%, and G2 in 41.2% and 41.2%, respectively. The ocular surface properties were worse in Group 1 than Group 2, and the difference was significant except for Schirmer's 1 test and IC. Conclusions Dry eye disorder is a complication of EKC and may cause a significant decrease in quality of life.

Visual rehabilitation by using corneal wavefront-guided transepithelial photorefractive keratectomy for corneal opacities after epidemic keratoconjunctivitis.

PURPOSE: To evaluate long-term visual and refractive outcomes of corneal wavefront-guided transepithelial photorefractive keratectomy (t-PRK) with mitomycin C for the treatment of corneal opacities secondary to adenoviral epidemic keratoconjunctivitis. METHODS: Records of patients who underwent corneal wavefront-guided t-PRK with excimer laser from January 2012 to December 2018 were retrospectively reviewed. Preoperative and postoperative uncorrected visual acuity, best-spectacle corrected visual acuity, slit-lamp biomicroscopic examination findings, manifest refraction, and corneal aberrations and fundus examination findings were evaluated. RESULTS: Twenty-two eyes of 22 patients comprising 12 male (55%) and 10 female (45%) were treated. The mean age was 34.5 ± 10.8 years (range 19-55). The mean follow-up time was 34.4 ± 17.50 months (range 13-61 months). There was a statistically significant improvement in UCVA and BSCVA (p < 0.001 and p = 0.02), and there was a significant decrease in total higher-order aberrations, spherical, coma and trefoil aberration at postoperative first year (p < 0.001 in each). In two eyes of two patients, minimal haze formation was observed after the procedure, and both eyes were treated with topical steroid. No recurrence was observed in subepithelial infiltrates in any patient during long-term follow-up. CONCLUSION: In long-term clinical follow-up, corneal wavefront-guided t-PRK treatment is an effective and reliable treatment method for rehabilitation of visual impairment due to corneal scars following adenoviral infections, in properly selected patients.

Pentacam corneal densitometry-guided treatment of adenoviral corneal subepithelial infiltrates: a comparative study between transepithelial phototherapeutic keratectomy and topical tacrolimus.

PURPOSE: To compare the changes in the measurement of corneal densitometry and total corneal higher-order aberrations (HOAs) between topical tacrolimus and transepithelial phototherapeutic keratectomy (Te-PTK) in the treatment of adenoviral corneal subepithelial infiltrates (SEIs). SUBJECT AND METHODS: This is an interventional prospective randomized study, including 63 eyes of 35 patients with symptomatic adenoviral corneal SEIs for at least 6 months. All patients underwent previous topical steroid therapy associated with unsatisfactory response and/or complications. Patients were assigned into three groups: (1) Te-PTK group: Te-PTK with MMC 0.02% was performed by a Technolas; Teneo excimer laser; (2) tacrolimus group: tacrolimus 0.03% ointment was applied once daily for 2-6 months; the endpoint of treatment was based on the improvement in the corneal densitometry, BCVA, and OSDI; and (3) control group: no intervention was done. BCVA, corneal densitometry, and total corneal higher-order aberrations (HOAs) evaluation using Pentacam HR were done at the baseline, 1 week, and then 1, 3, 6, and 12 months of the study. RESULTS: The study population was similar between all groups. The mean follow-up was 12.75 ± 0.9 months. Bilateral corneal SEI was recorded at 80%. At 12-month follow-ups, the mean BCVA improved in both the Te-PTK and tacrolimus groups without significant changes in the control group. The mean corneal densitometry of the anterior, central, and total cornea decreased significantly in the Te-PTK and tacrolimus groups. HO-RMS and total RMS decreased significantly in the Te-PTK and tacrolimus groups. BCVA, corneal densitometry (anterior, central, and total cornea), and corneal aberrations (total coma, total trefoil, HO-RMS, and total RMS) values were significantly better for the Te-PTK and tacrolimus groups than the control group. There were no statistically significant differences between Te-PTK and tacrolimus groups in terms of BCVA, corneal densitometry, corneal HOA, and the persistence of corneal SEIs. The persistence of corneal SEIs was significantly lower in Te-PTK and tacrolimus groups than the control group. CONCLUSION: Te-PTK and topical tacrolimus are effective methods for the treatment of adenoviral corneal SEIs improving visual acuity, corneal densitometry, and corneal HOA. The densitometry program of the Pentacam may give an objective guide for the treatment of adenoviral corneal SEIs. TRIAL REGISTRATION: ClinicalTrials.gov ID is NCT04267991.

Two cases of epidemic keratoconjunctivitis-associated dacryocystitis.

This report presents two cases of epidemic keratoconjunctivitis (EKC)-associated dacryocystitis. Both patients presented with preseptal edema. Orbital computed tomography (CT) scans were performed out of concern for orbital cellulitis and revealed soft tissue edema involving the preseptal aspect of the eyelids and also the lacrimal sac. Both patients were initially diagnosed with bacterial preseptal cellulitis with dacryocystitis and admitted for treatment with intravenous antibiotics. After no improvement on antibiotics, both patients were noted to have exam findings consistent with viral conjunctivitis, and one patient had a positive conjunctival swab for adenovirus. Both patients were subsequently treated with topical steroids, and symptoms improved. EKC has previously been identified as a cause of acquired nasolacrimal duct obstruction and canalicular stenosis, and acute inflammation of the lacrimal apparatus may explain this link. Dacryocystitis may also be contributory to the copious tearing seen in patients with epidemic keratoconjunctivitis.

An outbreak of epidemic keratoconjunctivitis caused by human adenovirus type 8 in primary school, southwest China.

BACKGROUND: Two outbreaks of epidemic keratoconjunctivitis (EKC) occurred successively with an interval of 5 days in two primary boarding schools in Weixi Lisu Autonomous County, Diqing, and Tibetan Autonomous Prefecture, Yunnan. The aims of this study were to determine the intensity and characteristics of the outbreaks, as well as the clinical manifestations in the patients, the risk factors for infection and the pathogen responsible for the two outbreaks. METHODS: An outbreak investigation was conducted in two primary schools, and a case-control study including patients from the Weixi County Ethnic Primary School was performed. Relevant specimens were collected according to the case definition, and next-generation sequencing was employed to identify the pathogen. An epidemiological investigation method was used to analyse the related epidemiological characteristics, such as risk factors. The phylogenetic tree was constructed by MEGA 7.0. RESULTS: A total of 331 acute conjunctivitis cases, including probable cases of EKC, were reported in the two schools, and the attack rates were 30.59% (171/559, 95%CI: 26.76-34.42) and 20.41% (160/784, 95%CI: 17.58-23.24), respectively. Cases occurred in all grades and classes in both schools, and only one staff member in each school presented illness. The epidemics lasted for 54 days and 45 days, respectively. The patients had typical manifestations of EKC, such as acute onset, follicular hyperplasia, pseudomembrane formation, preauricular lymphadenopathy, corneal involvement and blurred vision, and a relatively long disease course (average 9.40 days, longest 23 days and shortest 7 days). The risk factor for infection was close contact with a patient or personal items contaminated by a patient. The pathogen responsible for the outbreaks was HAdV-8. The virus was highly similar to the 2016 HAdV-8 strain from Tibet, China. CONCLUSIONS: This study strongly suggests that HAdV-8 could lead to serious consequences. This is the second report of a HAdV-8-associated EKC outbreak in mainland China. Tibetan HAdV-8 might be circulating in southwest China; therefore, it is necessary to monitor the pathogens causing acute conjunctivitis in this area.

A 8-year retrospective clinical analysis of 272 patients of epidemic Keratoconjunctivitis in Beijing, China.

BACKGROUND: Epidemic keratoconjunctivitis (EKC) is one of the most common eye infections worldwide. The analysis of clinical manifestations in different age groups help better know the disease. This study aims to provide more detailed analysis of 272 cases of EKC in 8 years, describe the differences of the clinical features among different age groups, and establish new clinical grading criteria. METHODS: 272 individuals were reviewed (2011-2019) in Beijing China. All the patients were classified into 3 grades according to the new grading criteria. The typical clinical signs of EKC and the photographs of the multiple subepithelial corneal infiltrates (MSI) were collected and analyzed. The number of 3 grades among and within different age groups were compared. The incidence of the typical signs among and within different age groups were compared. The proportion of each region of the cornea involved by MSI were compared. RESULTS: No significant differences were detected among the 4 groups in terms of the number of mild, moderate and severe cases, no matter in all-patients analysis (P = 0.271) nor in acute-phase-patients analysis (P = 0.203). The proportion of the severe cases was the highest among all patients (P = 0.000). Among the incidence of the typical signs, corneal involvement was the most common accounting for 69.8% (P<0.05). The probability of central region involvement was significantly higher than that of pericentral region involvement (P = 0.015) and peripheral region involvement (P = 0.000). CONCLUSIONS: Appropriate attention should be paid on EKC, because of the considerable proportion of severe cases, the high incidence of corneal lesion, and the high incidence of central region involvement of MSI.

Human Adenovirus Type 37 Uses αVß1 and α3ß1 Integrins for Infection of Human Corneal Cells.

Epidemic keratoconjunctivitis (EKC) is a severe, contagious ocular disease that affects 20 to 40 million individuals worldwide every year. EKC is mainly caused by six types of human adenovirus (HAdV): HAdV-8, -19, -37, -53, -54, and -56. Of these, HAdV-8, -19, and -37 use sialic acid-containing glycans as cellular receptors. αVß3, αVß5, and a few additional integrins facilitate entry and endosomal release of other HAdVs. With the exception of a few biochemical analyses indicating that HAdV-37 can interact physically with αVß5, little is known about the integrins used by EKC-causing HAdVs. Here, we investigated the overall integrin expression on human corneal cells and found expression of α2, α3, α6, αV, ß1, and ß4 subunits in human corneal in situ epithelium and/or in a human corneal epithelial (HCE) cell line but no or less accessible expression of α4, α5, ß3, or ß5. We also identified the integrins used by HAdV-37 through a series of binding and infection competition experiments and different biochemical approaches. Together, our data suggest that HAdV-37 uses αVß1 and α3ß1 integrins for infection of human corneal epithelial cells. Furthermore, to confirm the relevance of these integrins in the HAdV-37 life cycle, we developed a corneal multilayer tissue system and found that HAdV-37 infection correlated well with the patterns of αV, α3, and ß1 integrin expression. These results provide further insight into the tropism and pathogenesis of EKC-causing HAdVs and may be of importance for future development of new antiviral drugs.IMPORTANCE Keratitis is a hallmark of EKC, which is caused by six HAdV types (HAdV-8, -19, -37, -53, -54, and -56). HAdV-37 and some other HAdV types interact with integrin αVß5 in order to enter nonocular human cells. In this study, we found that αVß5 is not expressed on human corneal epithelial cells, thus proposing other host factors mediate corneal infection. Here, we first characterized integrin expression patterns on corneal tissue and corneal cells. Among the integrins identified, competition binding and infection experiments and biochemical assays pointed out αVß1 and α3ß1 to be of importance for HAdV-37 infection of corneal tissue. In the absence of a good animal model for EKC-causing HAdVs, we also developed an in vitro system with multilayer HCE cells and confirmed the relevance of the suggested integrins during HAdV-37 infection.

Sorting Motifs in the Cytoplasmic Tail of the Immunomodulatory E3/49K Protein of Species D Adenoviruses Modulate Cell Surface Expression and Ectodomain Shedding.

The E3 transcription unit of human species C adenoviruses (Ads) encodes immunomodulatory proteins that mediate direct protection of infected cells. Recently, we described a novel immunomodulatory function for E3/49K, an E3 protein uniquely expressed by species D Ads. E3/49K of Ad19a/Ad64, a serotype that causes epidemic keratokonjunctivitis, is synthesized as a highly glycosylated type I transmembrane protein that is subsequently cleaved, resulting in secretion of its large ectodomain (sec49K). sec49K binds to CD45 on leukocytes, impairing activation and functions of natural killer cells and T cells. E3/49K is localized in the Golgi/trans-Golgi network (TGN), in the early endosomes, and on the plasma membrane, yet the cellular compartment where E3/49K is cleaved and the protease involved remained elusive. Here we show that TGN-localized E3/49K comprises both newly synthesized and recycled molecules. Full-length E3/49K was not detected in late endosomes/lysosomes, but the C-terminal fragment accumulated in this compartment at late times of infection. Inhibitor studies showed that cleavage occurs in a post-TGN compartment and that lysosomotropic agents enhance secretion. Interestingly, the cytoplasmic tail of E3/49K contains two potential sorting motifs, YXXΦ (where Φ represents a bulky hydrophobic amino acid) and LL, that are important for binding the clathrin adaptor proteins AP-1 and AP-2in vitro Surprisingly, mutating the LL motif, either alone or together with YXXΦ, did not prevent proteolytic processing but increased cell surface expression and secretion. Upon brefeldin A treatment, cell surface expression was rapidly lost, even for mutants lacking all known endocytosis motifs. Together with immunofluorescence data, we propose a model for intracellular E3/49K transport whereby cleavage takes place on the cell surface by matrix metalloproteases.

Identification and molecular characterization of adenovirus types (HAdV-8, HAdV-37, HAdV-4, HAdV-3) in an epidemic of keratoconjunctivitis occurred in Pune, Maharashtra, Western India.

Epidemic keratoconjunctivitis (EKC) is a highly contagious infectious disease of the ocular surface and is caused mainly due to adenoviruses species D, B, and E. The present study was carried out to identify and characterize the viral etiological agents associated with the keratoconjunctivitis cases reported from Pune (Maharashtra), Western India between November-December 2013 and January, October-November 2014. Conjunctival swab specimens (n = 23) obtained from keratoconjunctivitis patients were subjected to detection of Adenovirus (AdV) and Enterovirus (EV) by PCR/RT-PCR using hexon and 5' NCR gene specific primers, respectively. Molecular typing of AdV and EV positive specimens was carried out by amplifying penton, fiber, and VP1 genes, respectively followed by sequencing and phylogenetic analysis. In this study, human adenovirus (HAdV) was identified as an etiological agent. None of the clinical specimens were found positive for enterovirus. AdV positivity in keratoconjunctivitis cases was found to be 60.9% (14/23). Fourteen of the HAdV positive strains, all of them were amplified by hexon gene, nine strains by fiber gene, and all 14 strains by penton gene specific primers. Sequencing of all HAdV positive samples revealed the presence of HAdV-8, HAdV-37, HAdV-3, and HAdV-4. All Indian strains showed highest nucleotide identity with the reference strains reported worldwide. The study revealed the circulation of HAdV-8 (78.6%) as predominant AdV strain followed by HAdV-37, HAdV-3, and HAdV-4 (7.2%) identified in the epidemic keratoconjunctivitis. Multiple types of AdVs in EKC reported for the first time in Western India. J. Med. Virol. 88:2100-2105, 2016. © 2016 Wiley Periodicals, Inc.

Resident corneal c-fms(+) macrophages and dendritic cells mediate early cellular infiltration in adenovirus keratitis.

The cornea contains a heterogeneous population of antigen-presenting cells with the capacity to contribute to immune responses. Adenovirus keratitis is a severe corneal infection with acute and chronic phases. The role of resident corneal antigen-presenting cells in adenovirus keratitis has not been studied. We utilized transgenic MaFIA mice in which c-fms expressing macrophages and dendritic cells can be induced to undergo apoptosis, in a mouse model of adenovirus keratitis. Clinical keratitis and recruitment of myeloperoxidase and CD45(+) cells were diminished in c-fms depleted, adenovirus infected mice, as compared to controls, consistent with a role for myeloid-lineage cells in adenovirus keratitis.

A unique secreted adenovirus E3 protein binds to the leukocyte common antigen CD45 and modulates leukocyte functions.

The E3 transcription unit of human adenoviruses (Ads) encodes immunomodulatory proteins. Interestingly, the size and composition of the E3 region differs considerably among Ad species, suggesting that distinct sets of immunomodulatory E3 proteins may influence their interaction with the human host and the disease pattern. However, to date, only common immune evasion functions of species C E3 proteins have been described. Here we report on the immunomodulatory activity of a species D-specific E3 protein, E3/49K. Unlike all other E3 proteins that act on infected cells, E3/49K seems to target uninfected cells. Initially synthesized as an 80- to 100-kDa type I transmembrane protein, E3/49K is subsequently cleaved, with the large ectodomain (sec49K) secreted. We found that purified sec49K exhibits specific binding to lymphoid cell lines and all primary leukocytes, but not to fibroblasts or epithelial cells. Consistent with this binding profile and the molecular mass, the sec49K receptor was identified as the cell surface protein tyrosine phosphatase CD45. Antibody-blocking studies suggested that sec49K binds to the membrane proximal domains present in all CD45 isoforms. Functional studies showed that sec49K can suppress the activation and cytotoxicity of natural killer cells as well as the activation, signaling, and cytokine production of T cells. Thus, we have discovered an adenovirus protein that is actively secreted and describe immunomodulatory activities of an E3 protein uniquely expressed by a single Ad species.

Structure-based design and optimization of potent inhibitors of the adenoviral protease.

Adenoviral infections are associated with a wide range of acute diseases, among which ocular viral conjunctivitis (EKC) and disseminated disease in immunocompromised patients. To date, no approved specific anti-adenoviral drug is available, but there is a growing need for an effective treatment of such infections. The adenoviral protease, adenain, plays a crucial role for the viral lifecycle and thus represents an attractive therapeutic target. Structure-guided design with the objective to depeptidize tetrapeptide nitrile 1 led to the novel chemotype 2. Optimization of scaffold 2 resulted in picomolar adenain inhibitors 3a and 3b. In addition, a complementary series of irreversible vinyl sulfone containing inhibitors were rationally designed, prepared and evaluated against adenoviral protease. High resolution X-ray co-crystal structures of representatives of each series proves the successful design of these inhibitors and provides an excellent basis for future medicinal chemistry optimization of these compounds.

Povidone iodine in the treatment of adenoviral conjunctivitis in infants.

CONTEXT: Adenoviral conjunctivitis. OBJECTIVE: To examine the effects of conjunctival irrigation with povidone iodine in infants with adenoviral conjunctivitis. MATERIAL AND METHODS: An outbreak of adenovirus conjunctivitis occurred between September and December 2012 at the neonatal intensive care unit of our hospital. Forty-three infants with adenoviral conjunctivitis were examined in this study. Thirty-five eyes of 35 infants who had detailed ophthalmological examination and clinical scoring were included. The conjunctiva of 15 infants (Group 1) were irrigated with povidone iodine 2.5% solution and then treated with preservative-free artificial tears and preservative-free antibiotic drops. Twenty infants (Group 2) were treated only with the same drops with the same posology, without conjunctival irrigation with povidone iodine. Patients were examined two times a week until the complete recovery. Lid edema, conjunctival chemosis, fragility of conjunctival vasculature, pseudomembrane formation, and corneal involvement were scored clinically. RESULTS: Twenty patients (57%) were female and 15 patients (43%) were male and mean age was 3.1 months (ranged from 1 to 4 months). No relation was found between gender and clinical scores. Statistically significant lower clinical scores were obtained from Group 1. The median recovery time was earlier in Group 1 (7 d) than Group 2 (12 d) (p = 0.001). CONCLUSION: Conjunctival irrigation with 2.5% povidone iodine is effective in the treatment of adenoviral conjunctivitis in infants.

Epidemiology of Keratoconjunctivitis Across India from 2017 to 2019: A Multicentric Hospital-Based Study.

PURPOSE: Conjunctivitis is one of the most common ocular conditions in clinical practice. Human adenoviruses have been the common causative agents known to cause epidemic kerato-conjunctivitis (EKC) in India from 1996 to 2019 with a positivity range of 13.8%-65.2%. The current study was initiated to throw light on the distribution of keratoconjunctivitis causing agents across India covering a span of 3 years. METHODS: A total of 709 swabs were collected from patients in viral transport medium (VTM), and real-time PCR was done to identify agents including Adenovirus (HAdV), Enterovirus, HSV, and Chlamydia. RESULTS: 47.8% of the samples were positive for HAdV followed by HSV (3.4%), Enterovirus (2.7%), and Chlamydia (0.6%). Overall, 386 people (54.4%) tested positive for one of these infections, with Chandigarh (88.4%) and Port Blair (71.7%) showing higher positivity rate. Pre-auricular lymphadenopathy and follicles were significantly associated with increased risk of conjunctivitis. CONCLUSION: Epidemiology of keratoconjunctivitis in the current study revealed HAdV to be predominant causative agent. Knowledge gained in such epidemiological studies guide us in outbreak expectations, limit antibiotic over-prescription, and enhance disease prevention.

Evaluation of anti-adenoviral effects of the polyvinyl alcohol iodine ophthalmic solution.

PURPOSE: To investigate the virucidal effects of a polyvinyl alcohol iodine, Saniode, against 16 types of human mastadenovirus (HAdV) causing ophthalmic, respiratory, gastrointestinal, urinary, and systemic infections. STUDY DESIGN: Laboratory investigation METHODS: Fifty microliters of Saniode were exposed to 10 µL each containing HAdV virus stock solution of 1 × 106 copies/µL of HAdV-1, -2, -3, -4, 5, -6, -7, -8, -11, -37, -53, -54, -56, -64, -81, and -85 for 10 s, 30 s, 1 min, and 3 min. After neutralization with 0.5% sodium thiosulfate, the mixture was diluted by ten-fold serial dilution and inoculated into 24 wells containing confluent A549 cell monolayers. Virucidal effects were calculated relative to the positive control on days 7-10 and observed until 30 days post-infection. RESULTS: Saniode satisfied the EN-14476 criterion for virucidal effects (>99.99%) for all HAdV types at all exposure times, including at 10 s on days 7 to 10 post-infection. All types of HAdVs that reacted for > 1 min achieved 99.99% reduction, including after 30 days. CONCLUSION: Saniode displayed virucidal effects against all tested HAdV types. Currently, with no specific medication available for HAdVs in ocular infection, this could be an option to prevent the spread of keratoconjunctivitis.

Relation Between Corneal Dendritic Cell Density and Tear Film Stability in Patients with Epidemic Keratoconjunctivitis Associated Dry Eye.

PURPOSE: To clarify the ocular surface features of patients with recent history of epidemic keratoconjunctivitis (EKC) and the relation between corneal dendritic cells (DCs) and ocular discomfort. METHODS: Normal controls (NC) and dry eye (DE) patients without EKC were recruited. Patients with recent EKC history (onset >4 weeks, but <20 weeks) were recruited as EKC + DE group (with dry eye) or EKC-DE group (without dry eye). Ocular surface disease index (OSDI) questionnaire, tear film parameters including lipid layer thickness, first tear break-up time (fBUT), average tear break-up time (aBUT), tear meniscus height and Schirmer I test, meibomian gland parameters, and in vivo corneal confocal microscopy were evaluated. RESULTS: 50 subjects in the NC group, 83 patients in the DE group, 76 patients in the EKC + DE group, and 38 patients in the EKC-DE group were included. Compared with the NC, DE, and EKC-DE groups, the EKC + DE group represented higher OSDI, lid margin, and meibum score (p < 0.05). In the EKC + DE group, the tear volume (10.5 ± 3.7 mm) was significantly higher than in the DE group (8.1 ± 2.8 mm, p < 0.001). The DC density in the EKC + DE group (29.98 ± 15.38 cells/image) was significantly higher than in NC, DE, and EKC-DE groups (4.68 ± 4.05 cells/image) (p < 0.001). The DC density was positively correlated with OSDI, lid margin, and meibum score (all p < 0.01) while inversely correlated with fBUT, aBUT (all p < 0.001) in the EKC + DE group. CONCLUSIONS: Corneal DC density significantly correlates to ocular discomfort and tear film instability in patients with recent EKC history who suffer from DE without aqueous tear deficiency.

Establishing an accurate and sensitive in vitro drug screening system for human adenovirus infection with human corneal cells.

Epidemic keratoconjunctivitis (EKC) is a hazardous and highly contagious disease, with the potential to cause epidemic outbreaks in hospitals and other community settings. There are currently no approved drugs for human adenovirus (HAdV), the causative agent of EKC. To establish a novel drug screening system for ocular HAdV infections, we employed CRL11516, a non-cancerous but immortalized human corneal epithelial cell line. Brincidoforvir and 3'-deoxy-3'-fluorothymidine inhibit replication of HAdV species C type 1 (C1), C2, E4, and C6 to the same extent. This alternative assay system may allow for the evaluation of anti-HAdV activity and cell cytotoxicity of compounds within 2 days and without the need of the rabbit eye infection model.

The molecular epidemiology, genotyping, and clinical manifestation of prevalent adenovirus infection during the epidemic keratoconjunctivitis, South of Iran.

PURPOSE: Adenoviral-mediated keratoconjunctivitis is among the emergency diseases of ophthalmology with long-term sequels. The role of adenovirus infection, ocular-related genotypes, and association with ocular symptoms need to be investigated for epidemiological as well as clinical purposes. METHODS: The affected patients from two close keratoconjunctivitis epidemics were included in the study. The swab samples were taken from patients; the total DNA was extracted and then used as a template for in-house Real-time PCR. Besides, partial Hexon genes of 11 adenovirus positive samples were amplified and submitted to sanger sequencing. Moreover, they were finally evaluated by phylogenetic analysis. RESULTS: Of 153 patients, 92 (60.1%) were males and 47 cases (30.7%) had a history of eye infection in the family or colleagues. Real-time PCR tests of 126 samples (82.4%) were positive for adenovirus, and all eleven cases that underwent sequencing analysis were determined to be group 8 (HAdV-D8). Adenovirus infection has a significant relationship with infection among family or colleagues (p = 0.048), membrane formation (p = 0.047), conjunctival bleeding (p = 0.046), tearing, and pain(p < 0.05). CONCLUSIONS: The results indicated that Adenovirus is the major cause of keratoconjunctivitis, and HAdV-D8 was the most common genotype in the area. There were some clinical manifestations associated with Adenovirus infection of the conjunctiva.