Abstract
Purpose: :
Human adenoviruses (HAdV) cause a wide array of infections, affecting the respiratory, gastrointestinal, genitourinary, and ocular surface mucosa. Identification (typing) of human adenoviruses in the last century relied solely on either serology or on limited sequencing of the adenoviral hexon gene, which contains the protein coding regions for the serologic determinant, and accounts for only 2.6% of the viral genome. Recently, whole genome analysis of emerging HAdVs has shown that either serum neutralization or limited sequencing of the hexon gene can lead to gross mischaracterization of an unknown adenovirus strain. As part of a larger project to sequence all the HAdVs within species D, the species associated most closely with epidemic keratoconjunctivitis, we describe our unexpected results of sequencing HAdV-D30.
Methods: :
HAdV-D30 was obtained from the American Type Culture Collection (ATCC, Manassas, VA), which identifies adenovirus strains by serum neutralization prior to archiving and commercial distribution. Virus was grown on A549 cells (ATCC) and purified by CsCl gradient, followed by high throughput sequencing to 17-fold or greater depth. Annotation was performed by a custom annotation engine with confirmation by two independent methods, and the results analyzed with mVISTA LAGAN, Simplot, and MEGA.
Results: :
Sequence assembly and analysis of the HAdV-D30 strain from ATCC revealed two distinct viral sequences, each with an identical hexon gene, also identical to the hexon gene previously archived for HAdV-D30. Subsequent pairwise alignment, phylogenetic, and recombination analysis revealed that one of the two viruses was a recombinant between HAdV-D30 and HAdV-D29, with the penton base and hexon genes of HAdV-D30 but the E3 transcription unit and fiber gene of HAdV-D29. Based on new adenovirus typing criteria at GenBank, the second virus was retyped as HAdV-D63.
Conclusions: :
The current availability of low cost, high throughput, genomic sequencing permits in depth analyses of the whole genomes of previously known as well as new adenovirus strains, and allows for much higher resolution, virus type determination. Exclusive reliance on serum neutralization can lead to mischaracterization of adenoviruses, and miss recombinant viruses, for example when two viruses with the same hexon gene occur in the same clinical sample. Whole genome sequencing remains the gold standard for proper characterization of HAdVs.
Keywords: adenovirus • keratitis • genetics