Molecular and Clinical Characterization of Human Adenovirus E4-Associated Conjunctivitis

Abstract

Purpose: To determine the characteristics of conjunctivitis associated with human adenovirus E4 (AdV E4).

Methods: Samples and outcomes from 500 patients with conjunctivitis were obtained from the NVC-422 randomized controlled clinical trial comparing auriclosene to placebo. Molecular typing identified 36 cases associated with AdV E4. Signs and symptoms at presentation and at the day 18 endpoint were compared with the larger cohort of 262 subjects with conjunctivitis caused by due to AdV D8. Full viral genomes of 22 AdV E4 isolates were reconstructed.

Results: AdV E4 was the most frequently identified adenoviral type in conjunctivitis cases from the United States. Signs and symptoms at presentation were comparable to those associated with AdV D8. Viral load at presentation was comparable between groups but resolution was more rapid in the AdV E4 group. Clinical signs were fully resolved by day 18 in 26 of 36 (72%) patients with AdV E4. Subepithelial infiltrates developed in 12 of 36 (33%) patients with AdV E4 compared with 98 of 215 (45%) patients with AdV D8 (P = .0001). One hundred twenty-four polymorphisms were observed among 22 whole viral genome sequences, which clustered into 3 clades. Patients in each clade developed subepithelial infiltrates. Neither single nucleotide polymorphism analysis nor machine learning approaches identified specific sequence features predictive of presenting signs or outcome.

Conclusions: AdV E4 conjunctivitis may be indistinguishable at presentation from AdV D8-associated disease. Resolution of viral load for AdV E4 appears more rapid than for AdV D8, and the risk for subepithelial infiltrates appears lower. Multiple substrains of AdV E4 are in circulation but all appeared equivalently pathogenic for conjunctivitis. NOTE: Publication of this article is sponsored by the American Ophthalmological Society.

Figure 1.

A. Presenting quantitative PCR-measured viral load of AdV E4 compared with AdV D8. Box represents mean ± one standard deviation, whiskers represent 25^th and 75^th percentiles. B. Timecourse of mean viral load in AdV E4 patients treated with NVC-422 (blue) or placebo (orange).

Figure 2.

Summary of 124 polymorphisms observed among 22 fully sequenced AdV E4 genomes. Top row show map of viral transcripts. Colored lines within grey bar show location of individual polymorphisms. Lower plot shows distribution of polymorphic loci among individuals, ordered by phylogenetic clustering.

Figure 3.

Jaccard distance similarity matrix for 22 full length AdV E4 sequences, clustered by phylip-generated phylogeny, demonstrating three clades of virus. Green = cases with subepithelial infiltrates, red = cases without.

Figure 4.

Distribution of polymorphisms among 75 AdV E4 samples (the 22 from the current study and 53 in GenBank database). Format as in Figure 2.

Figure 5.

Jaccard distance similarity matrix for 75 full length AdV E4 sequences shown in Figure 4, clustered by phylip-generated phylogeny, demonstrating four clades of virus.

Figure 6.

Concordance of phylogenetic clusters of AdV E4 in current study (left) and all AdV E4 isolates in GenBank (right). 3 of 4 clades identified in the larger group are represented among the 22 samples.

Figure 7.

Country of origin and clinical outcome among cases of AdV E4 conjunctivitis. Cases with pink or brown marker on outer circle are from present study; pink are cases without subepithelial infiltrates and brown are cases with subepithelial infiltrates. See legend for country of origin of isolates. Cases in white do not have specified country of origin in annotation in GenBank.

Figure 8.

‘Manhattan plot’ of association of individual single nucleotide polymorphisms of viral genome with development of subepithelial infiltrates. X-axis is position in viral genome, Y-axis is probability of association. Red dotted line is P value for 0.05 significance for uncorrected data; blue line is P value for significance after Bonferroni correction.