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Journal of Clinical Microbiology, October 2008, p. 3259-3269, Vol. 46, No. 10
0095-1137/08/$08.00+0     doi:10.1128/JCM.02354-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Epidemic Keratoconjunctivitis Due to the Novel Hexon-Chimeric-Intermediate 22,37/H8 Human Adenovirus

Koki Aoki,¹ Hiroaki Ishiko,^2* Tsunetada Konno,² Yasushi Shimada,² Akio Hayashi,² Hisatoshi Kaneko,³ Takeshi Ohguchi,¹ Yoshitsugu Tagawa,¹ Shigeaki Ohno,¹ and Shudo Yamazaki⁴

Department of Ophthalmology and Visual Sciences, Hokkaido University Graduate School of Medicine, Sapporo 060-8638, Japan,¹ Host Defense Laboratory, Mitsubishi Chemical Medience Corporation, Tokyo 174-8535, Japan,² Department of Microbiology, Fukushima Medical University School of Medicine, Fukushima 960-1295, Japan,³ AIDS Vaccine Development Association, Tokyo 169-0075, Japan⁴

Received 8 December 2007/ Returned for modification 5 March 2008/ Accepted 22 July 2008

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In a 2-month period in 2003, we encountered an outbreak of epidemic keratoconjunctivitis (EKC) in Japan. We detected 67 human adenoviruses (HAdVs) by PCR from eye swabs of patients with EKC at five eye clinics in different parts of Japan. Forty-one of the 67 HAdV DNAs from the swabs were identified as HAdV-37 by phylogenetic analysis using a partial hexon gene sequence. When the restriction patterns of these viral genomes were compared with that of the HAdV-37 prototype strain, one isolate showed a never-before-seen restriction pattern. Within 1 year, we encountered three more EKC cases caused by a genetically identical virus: two nosocomial infections at two different university hospitals and a sporadic infection at an eye clinic. We determined the nucleotide sequences of the full-length hexon and fiber genes of these isolates and compared them to those of the 51 prototype strains. Surprisingly, the sequence of the hexon ( determinant) loop-1 and -2 regions showed the highest nucleotide identity with HAdV-22, a rare EKC isolate. However, the nucleotide sequence of the fiber gene was identical to that of the HAdV-8 prototype strain. 22 We propose that this virus is a new hexon-chimeric intermediate HAdV-22,37/H8, and may be an etiological agent of EKC.

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Human adenoviruses (HAdVs) belong to the genus Mastadenovirus of the family Adenoviridae and infect billions of people worldwide, causing various diseases, including conjunctivitis, respiratory infectious disease, diarrhea in infants and young children, and hemorrhagic cystitis (31). Currently, 51 serotypes have been identified and reclassified into six species, HAdV-A to -F, on the basis of nucleotide (nt) and deduced amino acid sequences (8). Conjunctivitis due to AdV is caused mainly by HAdV-3 (in HAdV-B), -4 (in HAdV-E), -8, -19, and -37 (all in HAdV-D). Among these, HAdV-8, -19, and -37 cause more-severe epidemic keratoconjunctivitis (EKC) than the others (2-4). Serotyping of HAdVs is generally performed by virus isolation followed by a neutralization test (NT) and hemagglutination inhibition test (HI) using type-specific antiserum (29). To date, amplification of the genome by PCR and determination of the nucleotide sequences followed by phylogenetic analysis have become the general procedure for both the classification and identification of HAdVs (21, 22, 28). In 2003, we encountered an EKC outbreak in Japan (Infectious Agents Surveillance Report [Weekly reports of adenovirus isolation/detection from epidemic keratoconjunctivitis cases, 2001-2007; http://idsc.nih.go.jp/iasr/prompt/graph/ad3.gif]) and detected HAdV DNA in 67 swabs from EKC patients who visited five eye clinics in different parts of Japan. Among the 67 HAdVs, 41 were identified as HAdV-37 by a phylogenetic analysis based on the partial sequence of conserved region 4 (C4) in the hexon gene (22). When the restriction patterns of viral genomes of 10 representative isolates from the swabs were compared with that of the HAdV-37 prototype strain, one strain, C075/Matsuyama/2003, showed a different restriction pattern. This virus was isolated in Matsuyama city on Shikoku Island. During the same period, we observed a nosocomial infection due to genetically the same virus as C075/Matsuyama/2003 at a university hospital in Tokyo. In the following year (2004), we encountered nosocomial EKC due to HAdV-37 in six hospitals and HAdV-19a in one hospital. We also found one nosocomial infection caused by a virus genetically identical to C075/Matsuyama/2003 at a university hospital in Yamaguchi. A C075/Matsuyama/2003-like virus also caused a sporadic infection at an eye clinic in Fukui, in the western part of Japan in 2004.

In this study we determined the nucleotide sequences of the entire hexon and fiber genes of the C075/Matsuyama/2003-like isolates and compared them with those of the 51 prototype strains. The 350 bp of the partial hexon C4 sequence showed 99.7 to 100% identity with that of the HAdV-37 prototype strain, whereas hexon loops 1 (L-1) and 2 (L-2), which contain the NT epitope, showed 99.6 to 100% nucleotide identity to HAdV-22. Moreover, the fiber knob, responsible for cell tropism, has a nucleotide sequence identical with that of the HAdV-8 prototype strain, although the penton base has a nucleotide sequence identical to that of HAdV-37. Therefore, we propose that this virus is a novel hexon-chimeric intermediate HAdV containing hexon C4, hexon L-1, hexon L-2, fiber knob, and penton genes derived from the HAdV-37, -22, -8, and -37 serotypes, respectively. We propose that this chimeric virus, HAdV-22,37/H8, be identified as a new causative agent of EKC.

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Patients and sample collection. We observed EKC outbreaks throughout Japan between 2003 and 2004 (Weekly reports of adenovirus isolation/detection from epidemic keratoconjunctivitis cases, 2001-2007; http://idsc.nih.go.jp/iasr/prompt/graph/ad3.gif) and collected a total of 171 conjunctival swabs from the EKC patients (Table 1). In 2003, 67 swabs were collected from sporadic infections in five eye clinics from different parts of Japan: 12 swabs from Sapporo, 4 from Tokyo, 22 from Matsuyama, 21 from Kumamoto, and 8 from Itoman. Nine swabs were obtained from patients with nosocomial infections in Tokyo. In 2004, 93 swabs were obtained from six nosocomial outbreaks from different parts of Japan: 41 swabs from Fukushima, 6 from Kyoto, 23 from Kobe, 7 from Himeji, 7 from Yamaguchi, and 9 from Miyazaki. Two swabs were obtained from patients with sporadic infections in Fukui. All swabs were positive for the AdV antigen by a lateral-flow immunochromatography assay (Adeno-check; Santen Pharmaceutical Co., Ltd., Osaka, Japan).

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TABLE 1. Type identification of HAdV DNA detected in conjunctival swabs from patients with EKC by phylogenetic analysis

Viruses. One hundred microliters of the swab was added to A549 cells and incubated at 37°C. AdVs were identified by staining infected cells with an AdV-specific monoclonal antibody (Chemicon International, Temecula, CA). The isolates were further propagated in A549 cells without plaque purification for genome typing and sequence analyses of full-length hexon, fiber, and penton base genes. The swabs and isolates from patients were supplied by the Reference Center of Nosocomial Infection, Hokkaido University Graduate School of Medicine, Sapporo, Japan. The prototype strains of 51 HAdVs were obtained from the American Type Culture Collection and the National Institute of Infectious Diseases, Tokyo, Japan. These reference viruses were used directly for DNA extraction without further propagation.

Phylogeny-based classification using the partial hexon sequence. Viral DNA was directly extracted from 100 µl of each conjunctival swab using a Sumitest EX-R&D kit (Medical & Biological Laboratories Co., Ltd., Nagoya, Japan) according to the manufacturer's instructions. After drying, the extracted DNA was dissolved in 10 µl of TE buffer (10 mM Tris, pH 8.0, 1 mM EDTA). A 350-bp section of the hexon C4 nucleotide sequence was amplified as described previously (22). In brief, PCR was carried out in a LightCycler Quick System 330 (Roche) with a forward primer, AdnU-S'2 [nt 20743 to 20762; 5'-TTCCCCATGGC(A/T/C/G)CACAA(C/T)AC-3'], and a reverse primer, AdnU-A2 [nt 21274 to 21296; 5'-TGCC(T/G)(A/G)CTCAT(A/G)GGCTG(A/G)AAGTT-3']. The positions of the primers were numbered according to the complete nucleotide sequence of the HAdV-2 strain (GenBank accession no. J01917). The PCR protocol was as follows: 95°C for 10 min for the initial activation of FastStart Taq DNA polymerase and the denaturation of template DNA, followed by 45 cycles of amplification, each consisting of denaturation at 95°C for 10 s, annealing at 70°C for 10 s, and primer extension at 72°C for 25 s. The PCR products were separated on 3% agarose gel and purified with a QIAquick gel extraction kit (Qiagen, Valencia, CA). The nucleotide sequences of the hexon and fiber genes were amplified as described elsewhere (21). PCR was performed in separate rooms for each PCR step (pre-PCR, specimen preparation and template addition, and post-PCR) to prevent cross contamination. All samples were handled using different pipettes with aerosol-resistant tips. Negative-control samples were assayed in each PCR run (10). The number of copies of HAdV DNA in the clinical samples was calculated by a standard curve using pAd8hxn as the standard, as described elsewhere (22).

The nucleotide sequences of the PCR products were determined using a CEQ 2000XL DNA analysis system with a DyeTerminator cycle sequencing kit (Beckman Coulter, Fullerton, CA) and compared with those of all 51 serotypes using SINCA (Fujitsu Limited, Tokyo, Japan). The evolutionary distances were estimated using Kimura's two-parameter method (19), and unrooted phylogenetic trees were constructed using the neighbor-joining method (25). Bootstrap analyses were performed with 1,000 resamplings of the data sets (13). Similarity plots were generated using SimPlot (version 3.5.1) (20), and the similarities were calculated for each window of 200 nt by the Kimura two-parameter method (19), with a transition/transversion ratio of 2.0. The window was successively advanced along the genome alignment in 20-nt increments.

Genome typing. Viral DNA was extracted from the infected cells in a 75-cm² plastic flask using 3 ml of Hirt lysis solution (10 mM Tris, 1 mM EDTA, and 0.6% sodium dodecyl sulfate at pH 8.0) (16). Proteinase K was added at a final concentration of 50 µg/ml, and the samples were incubated at 37°C for 1 h. Cellular DNA was precipitated with 1 M NaCl (final concentration) overnight at 4°C. After phenol-chloroform extraction, the supernatant was treated with a mixture of ribonucleases A (25 mg/ml) and T1 (80 U/ml) (Sigma, St. Louis, MO), and phenol-chloroform extraction was performed. Viral DNA was precipitated with isopropanol and suspended in 300 µl of TE buffer. One microgram of viral genomic DNA was digested with 5 U of each of the restriction enzymes BamHI, BglI, BglII, HindIII, KpnI, SalI, XhoI, EcoRI, SacI, and SmaI (Takara Shuzo Co., Ltd., Kyoto, Japan). The digested viral DNA was loaded onto 1% agarose gels containing 1 µg/ml ethidium bromide. The DNA bands were photographed with an UV transilluminator and a Polaroid camera. The migration patterns of the DNA fragments were compared with those of previously reported genome types (1, 9, 15, 26, 30).

Serological analysis. Serological analyses were performed by a quantitative NT with type-specific antisera (HAdV-8, -19, -37) purchased from Denka Seiken Co., Ltd. (Tokyo, Japan) or from the American Type Culture Collection (HAdV-8). The antiserum against HAdV-22 was kindly supplied from the Hiroshima City Institute of Public Health. The NT was performed with A549 cells in 96-well microplates. The 100 50% tissue culture infective dose that caused a cytopathic effect after 7 days of incubation at 37°C was used for the challenge virus. Duplicates of the twofold-serially-diluted antisera were used in the NT (27).

Nucleotide sequence accession numbers. The GenBank accession numbers obtained in this study are AB369371 to AB369373 and AB437260 to AB437280. GenBank sequences AB330082 to AB330132 were used to generate alignments of the hexon gene.

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Phylogeny-based classification using a partial hexon C4 sequence. We detected HAdV DNA from 171 conjunctivitis patients whom we tested during 2003 to 2004. To assess the genetic constellation, an alignment of the 350-bp partial hexon C4 nucleotide sequence of the HAdV DNA was performed for the 51 prototype strains using the SINCA genetic software program. The 51 prototype strains showed 73.4 to 99.7% (average, 87.1%) identity, with the exception of HAdV-11, HAdV-35, HAdV-21, and HAdV-50 (data not shown).

Out of 67 sporadic infections in 2003, 58 sequences (86.6%) were segregated into cluster D, 4 into cluster E, and 5 into cluster B (Fig. 1). Cluster D consists of 32 serotypes, HAdV-8 to -10, -13, -15, -17, -19, -20, -22 to -30, -32, -33, -36 to -39, -42 to -49, and -51, whose nucleotide identities range from 92.3% (between HAdV-8 and HAdV-28) to 99.7% (between HAdV-24 and HAdV-38, HAdV-25 and HAdV-38, and HAdV-32 and HAdV-38), with an average of 97.3%. Of the 58 cluster D sequences, 41 (70.7%) presented 99.7 to 100% nucleotide identity to the HAdV-37 prototype strain, 4 sequences presented 100% nucleotide identity to HAdV-19a, and 13 sequences presented 99.7 to 100% nucleotide identity to another recently identified HAdV (17). All HAdV DNAs from nosocomial infections in Tokyo presented 100% nucleotide identity to HAdV-37. Eighty-seven of the 95 sequences (91.6%) from the patients in 2004 exhibited 99.4 to 100% nucleotide identity to HAdV-37, and 6 sequences exhibited identity to HAdV-19a (Fig. 1). Two HAdV DNAs from sporadic infections in Fukui showed 100% nucleotide identity to HAdV-37. These results showed that HAdV-37 was a major causative agent of EKC in 2003 to 2004.

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FIG. 1. Phylogenetic analyses of the representative strains C075/Matsuyama/2003, 8/Tokyo/2003, 1/Yamaguchi/2004, and FS-161/Fukui/2004 (all marked with *) from patients with EKC. A 350-bp partial sequence of the hexon gene of the representative samples was analyzed by the neighbor-joining method together with those of the prototype strains of all 51 HAdV serotypes. The numbers at the nodes are percentages of 1,000 bootstrap pseudoreplicates containing the cluster distal to the node.

Genome typing. To determine the genotype of representative HAdV isolates in cluster D from sporadic inflections in 2003, the isolates were propagated in A549 cells (Table 1). Clinical isolates were inoculated into A549 cells in 75-cm² plastic flasks. When the cytopathic effect was near completion, virus-infected cells were collected. Viral DNA was extracted from the infected cells as has been described elsewhere. The genomic DNA from the strain was digested with the 10 restriction endonucleases described in Materials and Methods. Interestingly, one strain, C075/Matsuyama/2003, isolated in Matsuyama City on Shikoku Island, showed a different restriction pattern from that of the HAdV-37 prototype strain in cluster D (Fig. 2). The genome size of C075/Matsuyama/2003 calculated by each fragment was approximately 3.5 kbp. These results suggested that the preparation did not contain a mixed virus population.

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FIG. 2. Genome type of the strain C075/Matsuyama/2003. The viral genomic DNA was digested with the restriction enzymes BamHI, BglI, BglII, HindIII, KpnI, SalI, XhoI, EcoRI, SacI, and SmaI. Lane 1, reference markers (EcoT14 and BglII digest of lambda DNA); lane 2, HAdV-37 prototype strain; lane 3, C001/Matsuyama/2003, which was identified as HAdV-37; lane 4, C075/Matsuyama/2003.

Full-length hexon gene nucleotide sequences. To clarify the discrepancy between the results of a phylogenetic analysis based on the partial hexon C4 region and genome typing, we amplified the full-length hexon gene (nt 17784 to 20618) of HAdV-37 using a pair of primers, AdVID (nt 17751 to 17772, 5'-TGTATGTGCCTTACCGCCAGAG-3') and AdL3D2 [nt 20642 to 20661, 5'-GCGC(A/T)CGATGG(A/G)CGC(A/G)AGCT-3'], and determined the full-length hexon nucleotide sequences. We also determined the full-length hexon nucleotide sequences of the 51 prototype strains. The nucleotide identity of the 51 prototype strains ranged from 65.6% (between HAdV-12 and HAdV-2) to 98.2% (between HAdV-15 and HAdV-29), with an average of 78.9%. Nineteen sequences from C075/Matsuyama/2003, two isolates (FS161/Fukui/2004 and FS165/Fukui/2004) from sporadic infections, and nine swabs (8/Tokyo/2003) and seven swabs (1/Yamaguchi/2004) from nosocomial infections had full-length hexon gene nucleotide sequences that were 99.4 to 100% identical. When full-length hexon genes of strains C075/Matsuyama/2003, FS161/Fukui/2004, FS165/Fukui/2004 (all from sporadic infections), 8/Tokyo/2003, and 1/Yamaguchi/2004 (both from nosocomial infections) were compared with those of the 51 serotypes, these five closely related strains showed only 89.6 to 89.7% nucleotide identity with HAdV-37, compared to 98.4% identity with the HAdV-22 prototype strain (data not shown). Based on the sequence analysis, the hexon gene was divided into four conserved (C1 to C4) and three variable (V1 to V3) regions (11). The nucleic sequence of the 51 prototype strains varies in length from 2,760 to 2,907 bp; however, the conserved regions have a constant length (2,283 bp, total). The sequences of C075/Matsuyama/2003, 8/Tokyo/2003, and 1/Yamaguchi/2004 had a length of 2,808 bp, identical to that of HAdV-22 but not to that of HAdV-37 (data not shown).

Recently, the nucleotide sequences of the hexon L-1 and L-2 regions, which contain the main NT determinant, were determined for all HAdV prototype strains, and criteria for typing was proposed (21). Therefore we compared the nucleotide sequences of the L-1 and L-2 regions of C075/Matsuyama/2003, 8/Tokyo/2003, 1/Yamaguchi/2004, and FS161/Fukui/2004 with those of the 51 prototype strains. The L-2 nucleotide sequence of the isolates showed the highest identity to HAdV-22 (99.6 to 100%) and only 77.0 to 77.3% nucleotide identity to HAdV-37 (Table 2). Therefore, the isolates formed a monophyletic cluster with HAdV-22, not with the HAdV-37 prototype strain (Fig. 3A). We also analyzed the L-1 region because of the higher contribution to the determinant (21). The isolates showed 99.4% nucleotide identity to HAdV-22 but 61.0% identity to HAdV-37 (Table 2). The isolates formed a monophyletic cluster with the HAdV-22 prototype strain (Fig. 3B).

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TABLE 2. Nucleotide sequence analysis of the L-1 and L-2 regions in the hexon gene and fiber knob regions

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FIG. 3. Phylogenetic analyses of the nucleic acid sequences of hexon L-2 (A), hexon L-1 (B), and the fiber knob (C). The nucleotide sequences of the strains C075/Matsuyama/2003, 8/Tokyo/2003, 1/Yamaguchi/2004, and FS161/Fukui/2004 (all marked with *) together with those of the 51 prototype strains of HAdV 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.

The inconsistent constellation of the loop region and the partial hexon C4 region suggested that recombination might have played an important role in the evolution of the isolates. To clarify the recombination events, we compared the full lengths of the hexon genes of strains C075/Matsuyama/2003 and 1/Yamaguchi/2004 with those of the 51 prototype strains and further analyzed them by similarity plot analysis with a sliding window of 200 residues (Fig. 4). When strain C075/Matsuyama/2003 was used as the query sequence and compared with the 51 prototype strains, its L-1 and L-2 regions were very close to those of the HAdV-22 prototype strain, whereas the hexon C4 region was similar to that of the HAdV-37 prototype strain (Fig. 4A). 1/Yamaguchi/2004 showed the same results as C075/Matsuyama (Fig. 4B). These results indicate that the hexon gene of the isolates is a chimera of HAdV-22 and -37.

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FIG. 4. Similarity plots of the nucleotide sequences of the full-length hexon gene calculated by SimPlot 3.5.1. The full lengths of the hexon nucleotide sequences of C075/Matsuyama/2003 (A) and 1/Yamaguchi/2004 (B) were compared with those of the 51 prototype strains. Each point represents the similarity in nucleotide sequence between the query strain and the 51 prototype strains, within a sliding window of 200 nt, centered on the positions plotted and with a step of 20 residues between points. Positions containing gaps were excluded from analysis. The vertical axis indicates the nucleotide identities between the isolates and the 51 prototype strains, expressed as percentages. The horizontal axis indicates the nucleotide positions of the hexon gene. The horizontal axis at the bottom indicates the positions of conserved regions (C1 to C4), variable regions (V1 to V3), and L-1 and -2.

Fiber and penton base nucleotide sequences. To date, several intermediate strains have been reported and are thought to have emerged by recombination events in the hexon and fiber genes of strains of different serotypes (12, 23). Therefore, we determined the nucleotide sequences of the full-length fiber genes of the isolates (C075/Matsuyama/2003, FS161/Fukui/2004) and strains from swabs (8/Tokyo/2003, 1/Yamaguchi/2004, FS161/Fukui/2004). The nucleotide sequence showed 99.9 to 100% identity with HAdV-8 but only 66.6 to 66.7% identity with HAdV-22 and 75.2 to 75.3% identity with HAdV-37 (data not shown). When the fiber knob nucleotide sequences of the isolates were compared with those of 51 prototype strains, the isolates showed 100% identity with HAdV-8 but only 59.2% identity with HAdV-22 and 83.9% identity with HAdV-37 (Table 2). This phylogenetic analysis showed the isolates forming a monophyletic cluster with the HAdV-8 prototype strain (Fig. 3C). The penton base nucleotide sequence of the isolates showed the highest identity (99.3%) with HAdV-37 (data not shown). Taken together, these results strongly support the idea that the isolates represent a hexon-chimeric intermediate AdV, HAdV- 22,37/H8, likely to be a new causative agent of EKC.

Serological analysis. To examine the serological reactivity between the C075/Matsuyama/2003 and HAdV-D strains, a quantitative neutralization assay was performed with the antiserum against HAdV-22 along with the antisera against the HAdV-8 and -37 prototype strains, which are causative agents of conjunctivitis. C075/Matsuyama/2003 reacted with HAdV-22 prototype-specific antiserum at a titer higher than 1:64 of the homologous titer. Conversely, it did not react with antisera specific for the HAdV-8 and -37 prototype strains. The low NT titer was found in the reaction only with HAdV-8 antiserum (Table 3). Consequently, the new serotype has been defined on the basis of its immunological distinctiveness, namely, a homologous neutralization titer/heterologous neutralization titer ratio of >16 in either direction (29). Therefore, these results suggest that the hexon L-1 and L-2 regions, which contain the main NT determinant of C075/Matsuyama/2003, are from HAdV-22.

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TABLE 3. Quantitative NT results with C075/Matsuyama/2003 against type-specific antisera

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The classical typing of AdVs is performed by NT and HI using type-specific antisera. In general, recombination events of different serotypes result in intermediate types with contradictory results by NT and HI. The molecular analysis of HAdVs has shown that the determinant in the hexon loop region is responsible for the neutralization properties, and the determinant in the fiber knob region is responsible for hemagglutination (14, 21, 24, 31). Therefore, to identify the intermediate strain by phylogenetic analysis, nucleotide sequence analysis of both the hexon loop region and the fiber knob region is required. An outbreak of EKC caused by a new intermediate AdV type 22/H8 has been reported in Germany (12). The hexon L-2 nucleotide sequence was identical to that of HAdV-22, and the fiber knob nucleotide sequence was identical to that of HAdV-8. This intermediate is very close to the one identified in this study.

In Japan, an intermediate HAdV has previously been isolated from patients with conjunctivitis and identified by NT and HI as a new causative agent of conjunctivitis, 22/H10,19,37 (23). The strain was kindly supplied from the Hiroshima City Institute of Public Health. To clarify whether HAdV-22/H10,19,37 is a fiber-chimeric virus, we determined the nucleotide sequences of the hexon L-2 region and the full-length sequence of the fiber genes and compared them with those of HAdV-10, -19, and -37. The L-2 region was identical to that of HAdV-22, whereas the nucleotide sequence of the full length of the fiber gene was identical to that of the HAdV-37 prototype strain (data not shown). The intermediate HAdV-22/H10,19,37 strain might be cross-reactive by HI using type-specific antisera against HAdV-10 and -19 because the nucleotide sequences of the fiber knob regions of HAdV-10, -19, and -37 are phylogenetically related (Fig. 3C). Phylogeny-based identification using the hexon L-1 and L-2 regions distinguished the 51 prototype strains by serotype. However, it did not correctly classify the 51 prototype strains into six species; e.g., HAdV-4 (in HAdV-E) was classified into HAdV-B (Fig. 3A and B), and HAdV-40 and -41 of HAdV-F were classified into HAdV-A (Fig. 3B). We have recently determined the partial hexon sequences of all 51 prototype strains and developed a rapid and reliable method for diagnosis based on phylogenetic analysis (22, 28). This method successfully classified the 51 prototype strains of HAdVs into the six designated species, as approved by the International Committee on Taxonomy of Viruses; we have applied molecular diagnosis to identify hundreds of isolates or swabs obtained over the last 30 years from patients with EKC and lower respiratory tract infections from different parts of the world (5, 6, 18, 22, 28). Through such study, we found another novel HAdV from nosocomially infected patients with EKC (17).

In Japan, we had four large outbreaks of EKC infections during the period from 1990 to 2001 and identified HAdV-37 as the causative agent by our methods (6). We also analyzed the genome types of HAdV-37 isolates and found five new genome types of HAdV-37. In 2003, we had an outbreak of EKC due to HAdV-37 again. When we analyzed the genome types of the representative isolates from each eye clinic, one isolate had a restriction pattern different from that of the HAdV-37 prototype strain. To clarify the discrepancy of our results, we amplified full-length hexon and fiber genes and compared their nucleotide sequences with those of the 51 prototype strains. The present study is the result of this investigation.

In conclusion, we here identified a new hexon-chimeric intermediate AdV, HAdV-22,37/H8, which was associated with sporadic infection and nosocomial infection in 2003 to 2004. This is, to our knowledge, the first report of a hexon-chimeric intermediate AdV causing an EKC outbreak. The origin and means of transmission of this strain are unknown. HAdV-22 was isolated from a patient with trachoma in 1956 (7); however, it is rarely isolated from patients with EKC. In Japan, we had an outbreak of EKC caused by HAdV-37 in 2003. It is well known that the determinant in the L-1 and L-2 regions is responsible for the neutralization properties. The ability to escape immunity to HAdV-37 may have been acquired by a recombination event. HAdV-22,37/H8 should be monitored as a likely new causative agent of EKC; 3 years after the period encompassed by this report (2003 to 2004), we additionally found an EKC nosocomial infection caused by this virus in Sapporo, in the northern part of Japan, in 2007 (data not shown). We recommend determining the hexon L-1 or L-2 region, partial C3 region, fiber knob, and penton base nucleotide sequences to precisely identify isolates.

 
We thank Noriko Inada (Division of Ophthalmology, Department of Visual Science, Nihon University School of Medicine) and Masako Nakamura (Fukui Prefectural Institute of Public Health and Environmental Science) for providing the swabs and isolates from EKC patients in Fukui prefecture.

 
* Corresponding author. Mailing address: Host Defense Laboratory, Mitsubishi Chemical Medience Corporation, Shimura 3-30-1, Itabashi-ku, Tokyo 174-8555, Japan. Phone: 81-3-5994-2340. Fax: 81-3-5994-2972. E-mail: Ishiko.Hiroaki{at}mp.medience.co.jp

Published ahead of print on 13 August 2008.

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Journal of Clinical Microbiology, October 2008, p. 3259-3269, Vol. 46, No. 10
0095-1137/08/$08.00+0     doi:10.1128/JCM.02354-07
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