Comparative analysis of ocular redness score evaluated automatically in glaucoma patients under different topical medications.

PURPOSE: To compare ocular redness score calculated automatically between glaucoma patients and healthy controls, and to assess the associations between this score and both demographical and clinical characteristics. METHODS: Glaucoma patients under different topical medications and matched controls were enrolled in this observational cross-sectional study. The Keratograph 5M (Oculus Optikgeräte GmbH) was used to automatically measure 5 redness scores: global; nasal bulbar; temporal bulbar; nasal limbal; temporal limbal. The Student t and ANOVA tests were used to compare continuous variables between groups. A multiple linear regression analysis was performed to evaluate the associations between redness scores and the use of different active principles. RESULTS: One hundred two glaucoma patients and 32 controls were included. Ocular redness scores were significantly higher in glaucoma patients compared to controls (always p < 0.001). The number of active principles was significantly associated with all the redness scores (always p < 0.05). The use of carbonic anhydrase inhibitors (CAIs) was the strongest predictor of overall redness, followed by prostaglandin analogs (PAs) and alpha-adrenergic agonists (AAAs) (respectively, ß = 0.400, p = 0.002; ß = 0.330, p = 0.013; ß = 0.311, p = 0.044). The post hoc analysis measuring the effect of different PAs on redness scores showed that overall redness and bulbar nasal redness scores were significantly lower in patients using tafluprost and latanoprost compared to those using travoprost and bimatoprost 0.01% (respectively, p = 0.025 and p = 0.024). CONCLUSION: Ocular redness was significantly higher in patients with glaucoma compared to control subjects. The number of active principles and the use of PAs, CAIs and AAAs were associated with higher redness scores.

Structural Elucidation of Viral Antagonism of Innate Immunity at the STAT1 Interface.

To evade immunity, many viruses express interferon antagonists that target STAT transcription factors as a major component of pathogenesis. Because of a lack of direct structural data, these interfaces are poorly understood. We report the structural analysis of full-length STAT1 binding to an interferon antagonist of a human pathogenic virus. The interface revealed by transferred cross-saturation NMR is complex, involving multiple regions in both the viral and cellular proteins. Molecular mapping analysis, combined with biophysical characterization and in vitro/in vivo functional assays, indicates that the interface is significant in disease caused by a pathogenic field-strain lyssavirus, with critical roles for contacts between the STAT1 coiled-coil/DNA-binding domains and specific regions within the viral protein. These data elucidate the potentially complex nature of IFN antagonist/STAT interactions, and the spatial relationship of protein interfaces that mediate immune evasion and replication, providing insight into how viruses can regulate these essential functions via single multifunctional proteins.

Genome-Wide Mutagenesis of Dengue Virus Reveals Plasticity of the NS1 Protein and Enables Generation of Infectious Tagged Reporter Viruses.

Dengue virus (DENV) is a major global pathogen that causes significant morbidity and mortality in tropical and subtropical areas worldwide. An improved understanding of the regions within the DENV genome and its encoded proteins that are required for the virus replication cycle will expedite the development of urgently required therapeutics and vaccines. We subjected an infectious DENV genome to unbiased insertional mutagenesis and used next-generation sequencing to identify sites that tolerate 15-nucleotide insertions during the virus replication cycle in hepatic cell culture. This revealed that the regions within capsid, NS1, and the 3' untranslated region were the most tolerant of insertions. In contrast, prM- and NS2A-encoding regions were largely intolerant of insertions. Notably, the multifunctional NS1 protein readily tolerated insertions in regions within the Wing, connector, and ß-ladder domains with minimal effects on viral RNA replication and infectious virus production. Using this information, we generated infectious reporter viruses, including a variant encoding the APEX2 electron microscopy tag in NS1 that uniquely enabled high-resolution imaging of its localization to the surface and interior of viral replication vesicles. In addition, we generated a tagged virus bearing an mScarlet fluorescent protein insertion in NS1 that, despite an impact on fitness, enabled live cell imaging of NS1 localization and traffic in infected cells. Overall, this genome-wide profile of DENV genome flexibility may be further dissected and exploited in reporter virus generation and antiviral strategies.IMPORTANCE Regions of genetic flexibility in viral genomes can be exploited in the generation of reporter virus tools and should arguably be avoided in antiviral drug and vaccine design. Here, we subjected the DENV genome to high-throughput insertional mutagenesis to identify regions of genetic flexibility and enable tagged reporter virus generation. In particular, the viral NS1 protein displayed remarkable tolerance of small insertions. This genetic flexibility enabled generation of several novel NS1-tagged reporter viruses, including an APEX2-tagged virus that we used in high-resolution imaging of NS1 localization in infected cells by electron microscopy. For the first time, this analysis revealed the localization of NS1 within viral replication factories known as "vesicle packets" (VPs), in addition to its acknowledged localization to the luminal surface of these VPs. Together, this genetic profile of DENV may be further refined and exploited in the identification of antiviral targets and the generation of reporter virus tools.