Adenoviral Pathogenesis in Epidemic Keratoconjunctivitis: the Failure of Hexon Gene Sequence to Predict Corneal Tropism

Abstract

Purpose: In the last century, human adenoviruses (HAdVs) were differentiated from one another by serum neutralization. This methodology depends on a stereotypical humoral immune response by the host to two adjacent hypervariable loops on the hexon protein, the most abundant adenovirus capsid component. Epidemic keratoconjunctivitis (EKC) is caused by HAdVs within species D, including HAdV-D56. Recent advances in genomic sequencing and bioinformatics led us to discover three other HAdVs with identical hexon genes to HAdV-D56.

Methods: Purified DNA from virus stocks were sequenced on a Roche 454 DNA sequencer by Operon to at least 17-fold depth, with an accuracy of greater than 99% (Q20 or better). The sequencing reads were assembled using CLC Genomics Workbench, with an N50 average of 5,260. Annotation was performed using a custom annotation engine. Open reading frames were BLAST-analyzed against GenBank sequences for confirmation and protein similarity. Splice sites were predicted using the GenScan web server at MIT. Phylogenetic analysis was performed using MEGA. mVISTA LAGAN was used for global pair-wise sequence alignment, and SimPlot and Bootscan were used to identify possible recombination events. Confocal microscopy with Cy3-labeled virus was used to identify differential entry into corneal epithelial and stromal cells in vitro.

Results: Four HAdV-Ds with a highly (>99%) identical hexon gene were identified, HAdV-D15, HAdV-D29, HAdV-D56, and a fourth previously untyped virus, which was identified from a second lot of HAdV-D15 (ATCC). All four viruses differed from one another across the remainder of their genomes. Homologous recombination was shown for the penton base RGD loop between the novel HAdV-D and HAdV-D53, another known EKC pathogen. By confocal microscopy, of the four viruses with the same hexon only HAdV-D56 readily entered both human corneal epithelial cells and fibroblasts.

Conclusions: Our findings show that the hexon gene hypervariable regions can be shared between otherwise disparate viruses, leading to mistyping if only partial genome sequencing is employed. These data further reinforce the importance of homologous recombination to the evolution of HAdV-Ds, including those viruses associated with EKC, and confirm an absence of contribution of the hexon to adenoviral tropism in the cornea.

Keywords: 411 adenovirus • 573 keratitis • 539 genetics