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.2012 May 14;53(6):2804-11.
doi: 10.1167/iovs.12-9656.

Analysis of human adenovirus type 19 associated with epidemic keratoconjunctivitis and its reclassification as adenovirus type 64

Affiliations

Analysis of human adenovirus type 19 associated with epidemic keratoconjunctivitis and its reclassification as adenovirus type 64

Xiaohong Zhou et al. Invest Ophthalmol Vis Sci..

Abstract

Purpose: Human adenovirus species D type 19 (HAdV-D19) has been associated with epidemic keratoconjunctivitis (EKC), a highly inflammatory infection of the ocular surface. Confusion exists regarding the origins of HAdV-D19. The prototype virus (HAdV-D19p) does not cause EKC, while a virus identified later with the identical serologic determinant is a significant ocular pathogen.

Methods: High throughput genome sequencing and bioinformatics analysis were performed on HAdV-D19p and three HAdV-D19 EKC strains, and compared to the previously sequenced clinical isolate, HAdV-D19 (C) and HAdV-D37. Corneas of C57BL/6J mice were injected with HAdV-D19p, HAdV-D19 (C), or virus-free buffer, and inflammation assessed by clinical examination, flow cytometry, and cytokine ELISA. Confocal microscopy and real-time PCR of infected corneal cell cultures were used to test viral entry.

Results: HAdV-D19 (C) and the other clinical EKC isolates showed nearly 100% sequence identity. EKC strains diverged from HAdV-D19p in the penton base, E3, and fiber transcription units. Simplot analysis showed recombination between EKC-associated HAdV-D19 with HAdV-D37, HAdV-D22, and HAdV-D19p, the latter contributing only the hexon gene, the principal serum neutralization determinant. HAdV-D19p induced stromal keratitis in the C57BL/6J mouse, but failed to infect productively human corneal epithelial cells. These data led to retyping of the clinical EKC isolates with a HAdV-D19 hexon gene as HAdV-D64.

Conclusions: HAdV-D19 associated with EKC (HAdV-D64) originated from a recombination between HAdV-D19p, HAdV-D37, and HAdV-D22, and was mischaracterized because of a shared hexon gene. HAdV-D19p is not infectious for corneal epithelial cells, thus explaining the lack of any association with keratitis.

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Conflict of interest statement

Disclosure:X. Zhou, None;C.M. Robinson, None;J. Rajaiya, None;S. Dehghan, None;D. Seto, None;M.S. Jones, None;D.W. Dyer, None;J. Chodosh, Alcon (C), Allergan (C)

Figures

Figure 1.
Figure 1.
Genomic and bioinformatics analysis of HAdV-D19. (A) Global pairwise alignment using mVISTA LAGAN compares HAdV-D19 (C) with HAdV-D19p and three clinical isolates of HAdV-D19 from patients with EKC. Transcription units are shown above the graph by black arrows relative to their position and orientation in the HAdV genome. Differences across the genome are seen when HAdV-D19 (C) is compared with HAdV-D19p, particularly in the penton base gene and E3 transcription unit. All three clinical EKC isolates were highly similar to HAdV-D19 (C). (B) Similarity plot analysis for HAdV-D19 (C) shows evidence for prior recombination with HAdV-D37, HAdV-D22, and HAdV-D19p. Note that only the hexon gene of HAdV-D19 (C) appears to have derived from HAdV-D19p. (C) Similarity plot analysis for HAdV-D37, another EKC pathogen, shows recombination with HAdV-D19 (C), and HAdV-D13, and confirms the plot shown in (B). (D) Whole genome phylogenetic analysis was performed for newly sequenced and previously archived HAdV-D19 strains, along with other HAdV-D genomes shown to be related closely by similarity plot analysis in (B) and (C) above. Bootstrap branches with values less than 70 were collapsed. HAdV-D19p claded separately from other HAdV-D19 strains.
Figure 2.
Figure 2.
HAdV-D19p in the mouse adenovirus keratitis model. (A) C57BL/6J mice injected intrastromally with 105 TCID of HAdV-D19 (C), HAdV-D19p, or virus-free dialysis buffer (mock) were compared at 4 days post infection for clinically evident keratitis. Representative photographs (n = 5 mice/group) show greater keratitis in the HAdV-D19p infected corneas. No clinically evident inflammation was observed in mock infected corneas. (B) Flow cytometric analysis of CD45+ cells in corneas (n = 4/group) removed from infected mice at 4 days post infection shows greater leukocyte infiltration in HAdV-D19p infected corneas when compared to HAdV-D19 (C) and mock infected corneas, and greater infiltration in HAdV-D19 (C) infected corneas than mock controls. (C) ELISA analysis (n = 4 mice/group) at 16 hours post infection shows greater CXCL1 expression in virus infected than in mock infected corneas, with no difference between viruses. However, IL-6 expression was greater in HAdV-D19p infected than HAdV-D19 (C) infected corneas or mock infected controls. (B,C) Data represent means of three independent experiments ± SD (*P < 0.05, ANOVA).
Figure 3.
Figure 3.
(A) Comparison of infection by Cy3-labeled HAdV-D19 (C) and HAdV-D19p in A549 cells, THE cells, and primary HCF, as seen by confocal microscopy at 30 minutes post infection. Virus appears red, while DAPI stains the cell nuclei blue, and phalloidin stains cellular actin green. Note that HAdV-D19p does not appear to infect THE cells (middle column, lower micrograph) at a time when HAdV-D19 (C) has already reached the perinuclear area (middle column, upper micrograph). (B) Assessment of adenoviral DNA by quantitative PCR, using primers against a conserved region of the E1B gene, as a measure of viral internalization and replication at one hour (1 H) and one week (1 W) post infection, respectively (n = 4/group). Comparing HAdV-D19 (C) and HAdV-D19p infection of all three cell types, it is apparent that HAdV-D19p fails to replicate (1 week post infection) in THE cells (middle graph), likely due to abortive viral entry. Data represent mean of three separate experiments ± SD (*P < 0.05; Student'st-test).
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