Identification of the Hexon and Fiber Gene of a New Strain of Adenovirus Involved in Epidemic Keratoconjunctivitis (EKC)

Abstract

Abstract: : Purpose: Adenovirus (Ad) is the common agent of keratoconjunctivitis. Ad3, Ad4, Ad7, Ad8, Ad19 and Ad37 account for 89% keratoconjunctivitis. An uncommon strain of adenovirus designated as Ad35/11 has been recovered from a patient with EKC. The hyper-variable regions (HVRs) of hexon and the fiber gene of Ad35/11 have been sequenced to find out possible variation that might be related to EKC. Methods: The identity of the isolate (Ad35/11) was determined by culture isolation and neutralization test (Culture-NT), and restriction endonuclease analysis (REA) of genomic DNA. Neutralization Test (NT) was performed on A549 cells in a 96-well microtiter plate. The antisera used were those for Ad3, -7, -11, -34, -35. For REA and sequence analysis, DNA was extracted from infected monolayer of A549 cells by standard method of phenol-chloroform extraction followed by ethanol precipitation. Genome typing was conducted with 10 restriction endonucleases following the manufacturer’s instruction. The fiber and the HVRs of hexon gene were sequenced with overlapping primers using a Genetic Analyzer 310 (Applied Biosystems). Nucleotide sequence of the isolate (Ad35/11) was compared with the other members of subgenus B and alignment was done with DNASIS software (Hitachi). Results: In NT the strain was neutralized by Ad35 and Ad11 antisera. Genomic DNA analysis with BamHI, HindIII, SalI , BglII, XbaI, XhoI, EcoRI, and BstEII showed restriction patterns similar to Ad11b and Ad35p. However, PstI and SmaI showed restriction pattern different from that of Ad11b and Ad35p. The overall homology of deduced amino acid (AA) of seven HVRs showed 90.4% and 74.0%, whereas overall fiber gene homology was 47.8% and 99.3% with Ad35p and Ad11p, respectively. The AA homology of the knob region of the fiber gene was 99.4% and 29.1%, homologous with Ad11p and Ad35p, respectively. Conclusions: In contrast with Ad11, Ad35 has not been reported so far as a causative agent of keratoconjunctivitis. The isolate is close to Ad35 in HVR of hexon and Ad11 in fiber gene. The HVRs of the hexon gene as well as the knob of the fiber to some extent carry neutralization epitope. Therefore, the isolate crossly reacted with both Ad35 and Ad11 in NT. Higher homology with Ad11 (99.4%) in the fiber gene might be related to the tropism for the eye.

Keywords: adenovirus • conjunctivitis • keratitis