An Outbreak of Epidemic Keratoconjunctivitis Caused by a New Intermediate Adenovirus 22/H8 Identified by Molecular Typing

Adenoviridae are nonenveloped, double-stranded DNA viruses with an icosahedral capsid. Human adenoviruses (HAdVs) are classified into 6 species (HAdV-A to HAdV-F) that consist of 51 HAdV types. Ophthalmologic manifestations of adenovirus infection include severe epidemic keratoconjunctivitis (EKC), which is almost exclusively caused by serotypes HAdV-D8, HAdV-D19, and HAdV-D37. Less severe pharyngoconjunctival fever is mainly caused by serotypes HAdV-B3, HAdV-B7, and HAdV-E4, and acute hemorrhagic conjunctivitis is mainly caused by serotype B11 [1].

Classical typing of adenoviruses is performed by neutralization tests using type-specific antiserum. The ɛ determinant in the hexon is responsible for the neutralization properties. Furthermore, typing can be performed by hemagglutination inhibition tests that reflect the properties of the hemagglutinin (γ determinant) located at the fiber knob [1]. More-rapid and more-precise molecular typing methods by sequencing have recently become available by systematic analysis of molecular phylogeny of the ɛ determinant [2].

In general, recombination events between different adenovirus types result in intermediate types with contradictory typing results in the neutralization and hemagglutination inhibition test. Intermediate types can also be identified by extending sequence analysis to the fiber knob (γ determinant). Recombination events may also result in new pathogenic properties and contribute to the generation of new adenovirus types—and even to the generation of new species exhibiting new patterns of organo-tropism and virulence [2].

HAdV-D22 was first isolated from a patient with trachoma in Saudi Arabia in 1956 [3] and is not usually reported as a causative agent of EKC. Furthermore, we never found HAdV-D22 in a systematic analysis of the circulation of human adenoviruses in Germany during the past 2 years, and the analysis included 119 clinical samples from patients with various diseases [4]. However, we cannot exclude the possibility that HAdV-D22 circulates in Germany and causes only mild symptoms and, therefore, is not being diagnosed. We report the first outbreak of EKC caused by HAdV-D22. Genetic analysis demonstrated an unusual recombination variant of HAdV-D22 with HAdV-D8 and HAdV-D37 that obviously resulted in high virulence.

Methods. Clinical and epidemiological information concerning the patients involved in the outbreak of EKC was collected retrospectively from the treating ophthalmologist and from the local health authorities. Adenovirus from 2 different patients was cultivated on A549 cells (isolates Hannover-2005-IAI-1 and Hannover-2005-IAI-2).

Diagnosis of adenoviral keratoconjunctivitis was confirmed by quantitative real-time PCR of conjunctival swab specimens [5]. HAdV DNA was amplified from clinical samples by a generic PCR protocol [6]. For typing purposes, the adenovirus hexon (ɛ determinant) loop-2 (L2) region from 2 outbreak patients was amplified and sequenced as described elsewhere [2]. After virus cultivation, the fiber knob region and the hexon loop-1 region was amplified as described elsewhere [2, 7]. Additionally, penton base sequencing was performed on the basis of a newly developed PCR protocol (authors' unpublished data). PCR products were separated in a 2% agarose gel (1% in the case of fiber amplicons) for 60 min at 120 V. DNA extraction from the agarose gels was performed with the Qiagen gel extraction kit (Qiagen), in accordance with the manufacturer's recommendations.

Both strands of PCR amplicons were cycle sequenced with rhodamine-labeled dideoxynucleotide chain terminators (DNA sequencing kit; ABI) and analyzed on an ABI Prism 310 automatic sequencer (Applied Biosystems). PCR primers were used for the sequencing reactions.

Two-step molecular typing was performed as described elsewhere [2]. A sequence diversity of <2.5% in the L2 region, compared with the next homologous prototype, was used as molecular typing criterion [2]. L2 sequences were compared with prototype sequences in GenBank by the Blast internet server.

Phylogenetic analysis was performed by using the Molecular Evolutionary Genetics Analysis (MEGA) software package, version 3.1 (Kumar, Tamura, Nei 2004). The phylogenetic trees were constructed with the neighbor-joining method (Kimura-2 parameter matrix), with a transition/transversion ratio of 2.0.

The following GenBank sequences were used to generate alignments of L2: HAdV-D8 (AB023546), HAdV-D9 (AF161562), HAdV-D10 (AB023548), HAdV-D17 (AF108105), HAdV-D19 (AF161565), HAdV-D22 (AJ745883), HAdV-D37 (AJ745892), and fiber knob region: HAdV-D8 (AB162771), HAdV-D9 (X74659), HAdV-D10 (AJ811442), HAdV-D17 (Y14241), HAdV-D19 (U69131), HAdV-D22 (AJ811445), and HAdV-D37 (U69132).

Results. The index patient received a diagnosis of keratoconjunctivitis at an ophthalmologic practice. Subsequently, within a period of 4 weeks, 11 other patients developed keratokonjunctivitis. Nine of these patients had been to the same ophthalmologic practice after the index patient for different medical reasons, and 2 were household contacts. Patients were 33–73 years old. Clinically, all patients presented with lid swelling, pain, photosenstitivity, and corneal subepithelial infiltrates. Two of the patients also presented with swelling of cervical lymph nodes, fever, and common cold symptoms evocative of pharyngoconjunctival fever.

Conjunctival swab specimens were obtained from 9 patients, and adenovirus was identified in all of them by quantitative real-time PCR [5]. of the 3 other patients, 2 lived in the same household as the virologically diagnosed patients, and no swab specimens were obtained, because diagnosis appeared to be clinically and epidemiologically evident. The source of infection for the index patient was not found. However, transmission in the ophthalmologic practice was obvious in the 8 patients presenting after the index patient, all of whom had been to the same ophthalmologic practice before onset of their symptoms. Cases in the other 2 patients are suspected to have been caused by intrafamilial transmission.

Viruses recovered from 2 patients (isolates Hannover-2005-IAI-1 and Hannover-2005-IAI-2) were analyzed by a 2-step molecular typing system [2]. Identification of species HAdV-D was achieved by sequencing of the amplicons of a generic hexon PCR [5] and identification of HAdV-D22 by sequencing of the amplicons of the highly variable L2 of the neutralization determinant ɛ. Both L2 sequences (accession numbers DQ404182 and DQ404185) showed 100% identity to each other and to the HAdV-D22 prototype sequences in the database (figure 1A). Additional sequencing of the loop-1 loop of the neutralization determinant ɛ (accession number DQ404181) confirmed L2 typing results. Results of neutralizations tests using a rabbit serum specific for HAdV-D22 confirmed the molecular typing results.

Figure 1

Phylogenetic analysis of nucleic acid sequences of the hexon loop 2 (A) and the fiber knob region (B) generated by the Neighbor Joining method, Kimura 2-parameter matrix (Molecular Evolutionary Genetics Analysis software package, version 3.1 [Molecular Evolutionary Genetics Analysis]).

To identify possible recombination events resulting in intermediate strains, the fiber knob region (γ determinant) of 2 patient isolates was also amplified and sequenced. The sequences of the fiber knob (DQ404183 and DQ404184) region showed 100% identity to HAdV-D8 sequences in the data bank (AB162771) (figure 1B) and displayed significant sequence divergence, compared with all other human adenovirus types. Therefore, the causative agent of the EKC outbreak was identified as a newly described intermediate adenovirus, type 22/H8.

Cell and organo tropism is determined by the fiber knob (γ determinant), as well as by the penton protein (RGD motif [arginine, glycine, and aspartic acid]); therefore, we extended sequence analysis to the penton gene. Penton base sequences of the 2 isolates (DQ404187 and DQ404186) were identical to penton base sequences of the EKC-associated HAdV-D37. This result indicates a second recombination event in the phylogenetic origin of the strains Hannover-2005-IAI-1 and Hannover-2005-IAI-2.

Discussion. EKC is typically reported to be caused by adenovirus types HAdV-D8, HAdV-D9, and HAdV-D37, which frequently cause nosocomial outbreaks [1]. Other adenovirus types have rarely been associated with EKC. This is the first description of an EKC outbreak caused by adenovirus type 22.

In contrast to types HAdV-D8, HAdV-D19, and HAdV-D37, HAdV-D22 is not clearly associated with keratoconjunctivitis, although it has some tropism for the eye. This type was first described in Saudi Arabia, and the first isolate came from the eye of a patient with trachoma in 1956 [3]. However, its association with trachoma was probably only coincidental. Since then, only 2 other reports mentioned HAdV-D22 with respect to ophthalmologic manifestations [8, 9]. The first report describes a laboratory infection caused by HAdV-D22 manifesting as follicular conjunctivitis without corneal involvement [8]. The second report describes a few cases of keratoconjunctivitis in Japan associated with HAdV-D22 without any evidence of an outbreak [9]. In the latter report, the virus had been typed by classic methods and DNA restriction-length polymorphism as an intermediate HAdV-D22 that had a hemagglutination determinant similar to HAdV-D10, HAdV-D19, or HAdV-D37. Because the hemagglutination determinants of HAdV-D10, HAdV-D19, and HAdV-D37 are phylogenetically closely related [2], it was probably impossible to get a more precise typing result by classical methods. However, the hemagglutination determinant of HAdV-D8 is phylogenetically distant from that of HAdV-D19 and HAdV-D37 (figure 1B). Therefore, the intermediate adenovirus type 22/H8, which we describe here as the agent responsible for the EKC outbreak, is not identical to the oculotropic HAdV-D22H10/19/37 described previously in Japan [9].

Because the fiber knob mediates the attachment of the virion to the primary receptor on the cell membrane [10], the ability of the intermediate type 22/H8 virus to cause keratoconjunctivitis is probably mediated by its fiber characteristics of type 8, an adenovirus type typically associated with EKC [1]. The hypothesis that the fiber characteristics are responsible for tropism is supported by a report that an adenovirus type 5 pseudotyped with the fiber of type 37 retains the ocular tropism of type 37 [11].

A second receptor for virus internalization are integrins-α_vβ3 and integrins-α_vβ5 in the cell membrane [12]. Their main ligand for binding is the RGD motif in the penton base of human adenoviruses [12]. Because interaction with integrins may also be important for cell tropism, we also sequenced the penton base of strains Hannover-2005-IAI-1 and Hannover-2005-IAI-2. Interestingly, these viruses had penton base sequences identical to that of HAdV-D37, which is also a typical causative agent of EKC. Therefore, our results indicate the significance of both penton base and fiber knob for cornea tropism of HAdV.

In conclusion, this is the first report of an EKC outbreak caused by an intermediate adenovirus type 22/H8 that may have acquired its unusual organo tropism and capability of epidemic spread by an additional recombination event with HAdV-D37. This recombinant virus may be a future cause of keratoconjunctivitis outbreaks. Further characterization of naturally occurring multiple recombinant HAdV isolates may enlighten our knowledge regarding organo tropism of HAdV and may also facilitate future design of highly specific gene therapy vectors.

• Received February 20, 2006.
• Accepted June 8, 2006.

• © 2006 by the Infectious Diseases Society of America