Molecular diagnosis of human adenoviruses d and e by a phylogeny-based classification method using a partial hexon sequence

Abstract

Human adenoviruses (HAdVs) are the major causes of a variety of acute illnesses. Virus isolation and neutralization tests are usually done to identify the causative virus, but these tests are labor-intensive and time-consuming, and standardized antisera are in limited supply. This study investigated a rapid and reliable method of virus identification based on PCR and phylogenetic analysis. The phylogenetic tree constructed by neighbor joining on the basis of the newly determined partial hexon sequences from 33 prototypes of HAdV-D and -E, along with 11 available prototypes of HAdV-A to -C and -F from GenBank, allowed HAdVs to be grouped into six distinct clusters. These clusters correspond closely to the six newly designated species, HAdV-A to -F. The partial hexon sequences of 57 isolates from patients with acute conjunctivitis obtained over 20 years plus those of 44 prototype strains were analyzed. Each isolate formed a monophyletic cluster along with its respective prototype strain, allowing serotype identification. Partial-hexon-based classification appears to be an effective tool for studying the molecular epidemiology of HAdVs.

FIG. 1.

Alignment of hexon genes from various AdVs. The primer sequences were aligned, along with available nucleotide sequences of AdVs from GenBank.

FIG. 2.

Phylogenetic analysis of AdV prototype strains and AdV-19a. The nucleotide sequences of a partial hexon region (916 bp) from 44 prototype strains and AdV-19a were analyzed by the neighbor-joining method. The numbers at the nodes are percentages of 1,000 bootstrap pseudoreplicates containing the cluster distal to the node.

FIG. 3.

Phylogenetic analysis of AdV serotypes 4, 8, 19a, and 37. Nucleotide sequences of a partial hexon region of the isolates, along with those of prototype strains and AdV-19a, were determined and analyzed by the neighbor-joining method. The numbers at nodes are percentages of 1,000 bootstrap pseudoreplicates containing the cluster distal to the node. Three AdV-4 isolates form cluster E (HAdV-E) with the AdV-4 prototype strain. Three AdV-8 isolates, two AdV-19a isolates, and three AdV-37 isolates form cluster D (HAdV-D) with their respective prototype strains.

FIG. 4.

Phylogenetic analysis of clinical isolates from patients with EKC. The results were obtained by the neighbor-joining method. The numbers at the nodes are percentages of 1,000 bootstrap pseudoreplicates containing the cluster distal to the node. The representative unidentified 5212/95* isolates formed a distinct cluster with the prototype strains of AdV-8 (Trim).