Predicting the Next Corneal Pathogen: Systems Biology Analysis of a Novel Human Adenovirus

Abstract

Purpose: : Whole genome sequencing and bioinformatics analysis recently identified several novel human adenoviruses (HAdVs), and demonstrated homologous recombination as an important mechanism in HAdV evolution. New models of ocular infection enable comprehensive testing of candidate ocular pathogens. We examined a new, previously unreported adenovirus, cultured from a child with respiratory infection, to determine the potential for a systems biology approach to identify possible future ocular pathogens.

Methods: : Genomic DNA from the HAdV isolate was Sanger sequenced. MEGA was used to construct phylogenetic trees, mVISTA LAGAN for global pair-wise sequence alignment, and Simplot for bootscan analysis. Immortalized human corneal and conjunctival epithelial cells, and primary human keratocytes, were infected with the viral isolate or HAdV-D37, a known and important corneal pathogen, and subjected to real-time RT-PCR for viral gene expression, viral titer for viral replication, and Western blot for Src phosphorylation. C57BL/6j mice were infected with purified viral isolate or HAdV-D37. ELISA and flow cytometry were performed on mouse corneas at 16 hrs and 4 days post infection (pi) to compare production of inflammatory mediators and CD45+ cell infiltration, respectively.

Results: : Whole genome sequencing and phylogenomics placed the viral isolate within HAdV species D, with similarity to HAdV-D49, and showed probable recombination with HAdV-D37 in the penton base gene. Confocal microscopy using Cy-3 labeled virus revealed viral entry into immortalized human corneal and conjunctival epithelial cells within 30 mins pi. RT-PCR confirmed viral gene expression in these cells equivalent to HAdV-D37. Src phosphorylation in infected keratocytes appeared greater than after infection with HAdV-D37. In the mouse adenovirus keratitis model, the new viral isolate was 100-fold more pathogenic than HAdV-D37, as confirmed by flow cytometry. ELISA revealed 6-fold more CXCL1 and 17-fold more IL-6 after infection than with HAdV-D37 at the same infectious dose.

Conclusions: : The new HAdV-D isolate is highly pathogenic in adenovirus keratitis models, suggesting its potential to cause serious ocular infections in humans. Our study is presented as a model for future systems biology approaches to predicting disease by new adenovirus pathogens.

Keywords: adenovirus • keratitis • inflammation