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Department of Ophthalmology and Visual Sciences, Hokkaido University Graduate School of Medicine
Sapporo City Institute of Public Health, Sapporo, Hokkaido
Research and Development Department, Mitsubishi Kagaku Bio-Clinical Laboratories, Inc., Itabashi-ku, Tokyo, Japan
ABSTRACT
Human adenovirus type 37 (HAdV-37) is a major cause of epidemic keratoconjunctivitis and has recently been the largest causative agent of keratoconjunctivitis in Japan. To investigate the genetic characteristics of HAdV-37 strains isolated in Sapporo, we analyzed the genome types and genetic relationships of 51 strains isolated there from 1990 through 2001. By using DNA restriction analysis, eight genome types (HAdV-37/D1, HAdV-37/D3, and HAdV-37/D6 to HAdV-37/D11) were identified, including five new ones. The restriction fragments of these genome types shared more than 95% identity with those of the prototype strain. By DNA sequence analysis, five and three single nucleotide substitutions, respectively, were found in partial sequences of the hexon and fiber genes. The combinations of mutations resulted in four hexon and fiber types (hx1 to hx4 and f1 to f4) and six hexon/fiber pairs (hx1/f1, hx2/f1, hx1/f2, hx1/f3, hx3/f4, and hx4/f4). The six pairs correlated well with certain genome types. In all three epidemics of keratoconjunctivitis to strike Sapporo in the past 12 years, specific genome types and fiber types were usually isolated: in the first epidemic, HAdV-37/D1 (f1) and HAdV-37/D3 (f1); in the second, HAdV-37/D6 (f2) and HAdV-37/D8 (f3); and in the third, HAdV-37/D10 (f4) and HAdV-37/D11 (f4). We conclude that mutations in the adenovirus genome occurred chronologically and that certain mutations were correlated with the epidemics of adenoviral keratoconjunctivitis.
INTRODUCTION
The human adenoviruses (HAdVs) in the genus Mastadenovirus of the family Adenoviridae form a group that includes 51 serotypes (11, 27). These were divided into six subgenera (A to F) according to their biologic, immunologic, and biochemical properties (8, 32). In 1999, reclassification of HAdVs on the basis of nucleotide and deduced amino acid sequences was approved by the International Committee on Taxonomy of Viruses, and consequently the 51 serotypes of HAdVs in the genus Mastadenovirus were grouped into six species, HAdV-A to HAdV-F (31). Adenoviral conjunctivitis is caused mainly by HAdV-3 (in HAdV-B), HAdV-4 (in HAdV-E), and HAdV-8, HAdV-19, and HAdV-37 (in HAdV-D). Among these, HAdV-8, HAdV-19, and HAdV-37 cause more severe conjunctivitis than the others (26). HAdV-37 was first isolated from patients with keratoconjunctivitis in 1976 in The Netherlands and was reported by De Jong et al. as a newly identified serotype (12). Ever since, HAdV-37 has been recognized as an important causative agent of keratoconjunctivitis (5, 12, 17). In Sapporo, in northern Japan, HAdV-37 has been isolated every year since 1977 (3) and has caused three large epidemics of conjunctivitis between 1990 and 2001 (Fig. 1) (25).
Restriction endonucleases provide an important instrument for epidemiological studies of adenoviruses. After HAdV-37 was initially characterized (33), many reports appeared about variations in the HAdV-37 genome isolated from patients with ocular and urethral diseases (2, 13, 14, 21, 22, 26). Reports in the 1980s showed that the genome of HAdV-37 strains seemed to be rather stable (2, 14, 21). In Sapporo, the genome type of HAdV-37 isolates was almost all HAdV-37p (4, 13, 14) until 1988. In Hiroshima, in western Japan, a genomic variant of HAdV-37, called HAdV-37a, was isolated between 1983 and 1986 (22). Since 1989, however, very few reports about HAdV-37 genome types have appeared. In other serotypes, genome type analysis and long-term surveillance have been actively pursued (1, 10, 15, 16, 20). The resulting reports suggested an association between clinical epidemiology and changes in genome types. Nevertheless, for HAdV-37 no reports have described a transition of genome types in at least 10 years. Nor have any reports examined the variability of nucleotide sequences of HAdV-37 variants.
In this study, we analyzed the annual transition of predominant HAdV-37 genome types isolated between 1990 and 2001 in Sapporo, Japan, and compared this transition with the transitions of nucleotide sequences of partial hexon genes and full-length fiber genes.
MATERIALS AND METHODS
Virus strains. Fifty-one isolates of HAdV-37, obtained from conjunctival swabs from the same number of patients with acute conjunctivitis, were used in this study. All clinical strains were isolated in Sapporo, in northern Japan. The years and numbers of the isolates are shown in Table 1. Among the 51 isolates, 46 strains between 1990 and 1999 were isolated in the Sapporo City Institute of Public Health. The other five strains were isolated in 2001 in a commercial clinical laboratory and then transported to Hokkaido University. All isolates were propagated in HeLa, A549, or Hep-2 cells and were identified as HAdV-37 by a neutralization method with serotype-specific antiserum purchased from Denka Seiken Co., Ltd. (Tokyo, Japan). A HAdV-37 prototype strain was purchased from the American Type Culture Collection (Manassas, Va.).
Viral DNA extraction. Viral DNA was extracted from the HAdV-infected cells in 75-cm2 plastic flasks as described by Shinagawa et al. with some modifications (30). The infected cells were suspended in 2 ml of lysis buffer (10 mM Tris, 10 mM EDTA, 1% sodium dodecyl sulfate at pH 8.0). Almost all cellular DNA and RNA were precipitated with NaCl (1 mol/liter) overnight at 4°C and discarded. The supernatant was extracted with an equal volume of phenol. Viral DNA was precipitated with 1.5 volumes of 100% ethanol added to the phenol phase including the interphase. After proteinase K treatment, viral DNA was extracted by phenol-chloroform extraction and precipitated with ethanol.
DNA restriction analysis. Aliquots of 1 μg of viral DNA were digested with 5 U of the following restriction endonucleases: BamHI, BglI, BglII, EcoRI, HindIII, SacI, SmaI, and XhoI (Takara Shuzo, Kyoto, Japan). The digested viral DNA was loaded onto 1.5% agarose gels containing 0.1 μg of ethidium bromide per ml and a 2-log DNA ladder marker (New England Biolabs, Beverly, Mass.). DNA bands were photographed with a UV transilluminator and a Polaroid camera, and the patterns of fragments were compared with previously reported patterns of HAdV-37 variants and the prototype strain.
PCR. A 956-bp conserved region of each hexon gene and the entirety of each fiber gene were amplified by the PCR method. The partial hexon genes were amplified as described by Saitoh-Inagawa et al. (24). Briefly, the primers for PCR amplification were AdnU-S' and AdnU-A. The PCR was carried out by using a Gene Amp PCR System 9600 (Applied Biosystems, Foster City, Calif.) by using a cycle consisting of 94°C for 1 min, 50°C for 1 min, and 72°C for 2 min, for a total of 36 cycles. After the last cycle, the DNA was extended at 72°C for 7 min. The fiber genes were amplified as follows. The primers AdD1 (5'-GATGTCAAATTCCTGGTCCAC-3', nucleotides [nt] 5 to 25 of GenBank accession number U69132) and AdD2 (5'-TACCCGTGCTGGTGTAAAAATC-3', nt 1196 to 1217 of GenBank accession number U69132) were used to amplify the entire open reading frame of HAdV-37 fiber genes (34). PCR was performed in 50-μl volumes containing 5 μl of 10x PCR buffer, a 0.2 mM concentration of each of deoxynucleoside triphosphate (i.e., dATP, dGTP, dCTP, and dTTP), a 0.5 μM concentration of each primer, 1 U of FS Taq DNA polymerase (Applied Biosystems), and 5 μl of DNA extract. The amplification reaction was carried out as described above.
Cycle sequence and nucleotide sequence analyses. After the PCR products were purified by a QIA-quick gel extraction kit (QIAGEN, Valencia, Calif.), the nucleotide sequences of the partial hexon genes and the entire fiber genes were determined with a BigDye Terminator Cycle Sequencing Ready Reaction Kit (Applied Biosystems) and with a 373A DNA auto sequencer (Applied Biosystems). The primers used for cycle sequencing were the same as those used in the PCR described above. An internal forward primer, Ad37Hxs (5'-GTGGCCCAATGCAACATGAC-3', nt 389 to 408 of GenBank accession number AB099368), and a reverse primer, Ad37Hxa (5'-CCCTGGTAGCCGATGTTGTA-3', nt 443 to 462 of GenBank accession number AB099368), were also used to sequence the hexon genes. For the fiber genes, two internal forward primers, Ad37fs1 (5'-TTAACAGGAAAAGGAATAGG-3', nt 455 to 474 of GenBank accession number U69132) and Ad37fs2 (5'-AAAGCAATTGGTTTTATGCC-3', nt 893 to 912 of GenBank accession number U69132), and one reverse primer, Ad37fa3 (5'-GATGTGTCTGGTGTTGTCCA-3', nt 608 to 627 of GenBank accession number U69132), were used. Cycle sequencing was performed under the conditions specified by the manufacturer (Applied Biosystems). The sequences of hexon and fiber from the isolates were analyzed, along with previously published fragments of HAdV-19p (hexon, AB099354; fiber, U69130), HAdV-19a (hexon, AB099384), and HAdV-37p (hexon, AB099368; fiber, U69132). We estimated the evolutionary distances by using the Kimura two-parameter method (19) and constructed unrooted phylogenetic trees by using the neighbor-joining method (23) by SINCA software (Fujitsu Ltd., Tokyo, Japan).
Nucleotide sequence accession numbers. The GenBank accession numbers of the nucleotide sequences newly identified in this study are AB161032 to AB161036.
RESULTS
DNA restriction analysis. At present, the nomenclature systems of genomic variants of HAdV-37 differ among the various reports. Also, the restriction endonucleases used for classifying genome type differ from study to study. For example, the genome type HAdV-37a named by Noda et al. (22) exhibits the same restriction pattern as that of HAdV-37d named by Guo et al. (13) as well as that of HAdV-37/D3 named by Adrian et al. (2). We intended that the restriction enzyme map in this study should correspond with the previously reported genome types and selected the same eight restriction enzymes and nomenclature system as selected in the report of Adrian et al. (2). According to this system, with which it is possible to classify all previously reported genome types, the strains representing the same restriction patterns as those of the prototype strain were named type D1. The D1 restriction patterns were coded number 1, and the various other patterns were consecutively numbered in order of chronological appearance. The genomic variants were represented by a combination of the code numbers of the eight restriction enzymes and were named D2, D3, etc., also in order of chronological appearance. In this way, Adrian et al. reported three restriction patterns with HindIII (enzyme codes 1 to 3) and one pattern with XhoI. Using the other six enzymes (BamHI, BglI, BglII, EcoRI, SacI, and SmaI), they reported two restriction patterns (2). They then identified five variants of HAdV-37, D1 to D5, according to their combinations of enzyme codes. In this study, we named the new HAdV-37 variants D6 to D11.
The 51 isolates of HAdV-37 analyzed in this study were divided into eight genome types according to the profile of restriction patterns (Table 2). Nine strains isolated between 1990 and 1993 were classified into genome type D1, and two strains isolated in 1991, along with one isolated in 1998, were classified into D3. The other 39 strains exhibited restriction patterns different from those of D1 to D5 and were divided into six genome types (D6 to D11) in order of chronological appearance. Among these six, genome type D8 exhibited the same patterns as the HAdV-37 variants reported by Kikuchi et al. (18). Thus, five new genome types were identified in this study. In this study, the restriction patterns created by BamHI, BglII, and Hind III were all identical with those of the prototype strain (code 1), and the enzyme code of EcoRI was identical with that of code 2 except for strain D1. Within types D6 to D11, the patterns of BglI, SacI, SmaI, and XhoI differed from those described previously. Specifically, as indicated in Fig. 2, types D6, D8, and D11 represented new BglI patterns (code 3). Moreover, D7 and D9 each represented still another pattern, and we coded them as 4 and 5. In the SacI profile, D10 and D11 represented a new pattern, code 3. In SmaI, D9 represented a new pattern, which we placed in code 3, while in XhoI, D8 and D9 represented a new restriction pattern, which we placed in code 2.
Among the 39 strains classified into types D6 to D11, D6 was the most commonly identified genome type. Eighteen strains isolated from 1990 to 1997 were classified as D6. Although only two strains of D7 were isolated, both in 1992, these were the only strains identified that year. Eleven strains of D8 were isolated between 1995 and 1997. Only one strain of D9 was isolated (1997). Types D10 and D11 have emerged since 1999; since that year, four D10 and three D11 strains have been isolated (Table 3).
Nucleotide sequence analysis. To analyze the relationship between each genome type in detail, we sequenced partial hexon genes and full-length fiber genes. Four types of nucleotide sequences were detected in the partial hexon genes. These were named hx1 to hx4 (Table 4). The first type, hx1, contained the strains of genome types D3, D6, D7, D8, and D9. HAdV-37p belongs to type hx1. The second, hx2, contained the strains of D1. Interestingly, although the restriction patterns of the D1 strains were identical with those of the prototype strain, the sequences of the partial hexon genes were different. The third type, hx3, contained the strains of type D10. The last type (hx4) contained the strains of D11. As shown in Table 4, hx2, hx3, and hx4 exhibited one, four, and five point mutations, respectively, compared with the sequence of the prototype strain. However, none of the mutations resulted in any amino acid substitutions. In the previous study, the phylogenetic analysis based on the partial hexon sequence was reported to be useful for the classification of adenoviral isolates (29). We therefore constructed a phylogenetic tree based on the partial hexon genes of HAdV-37 (prototype and hx1 to hx4) with those of HAdV-19p and HadV-19a (another major causative HAdV-D of adenoviral keratoconjunctivitis). As shown in Fig. 3, HAdV-37p and HAdV-37 isolates (hx1 to hx4) formed a distinct cluster separated from HAdV-19p and HAdV-19a. In the cluster of HAdV-37, four hexon sequence types were divided into two clusters; one cluster contained hx1 and hx2, and the other included hx3 and hx4 (Fig. 3). Since the partial hexon sequences of HAdV-19p and HAdV-19a were quite different from each other, they did not form a distinct cluster (9, 29).
Likewise, the fiber gene sequences were divided into four clusters, f1 to f4 (Table 5). Cluster f1 contained the strains of genome types D1, D3, and D7. The only strain of genome type D6 (isolated in 1990) was included in f1. The nucleotide sequence of f1 was the same as that of the prototype strain. Cluster f2 contained the strains of D6 isolated after 1993. This cluster exhibited one point mutation, at position 270 on the fiber gene of HAdV-37p. This point mutation caused a Gly-to-Glu substitution. Cluster f3 contained the strains of D8 and D9 and exhibited two point mutations. One was the same as f2, and the other was localized to position 701 on the fiber genes. However, this latter point mutation did not result in amino acid substitutions. The last cluster, f4, contained the strains of types D10 and D11. This cluster exhibited one point mutation, which differed from those of f2 and f3; it was from G to C at position 350 and resulted in a Glu-to-Gln amino acid substitution. In the phylogenetic tree based on the fiber gene sequences, all fiber gene sequences of HAdV-37 isolates formed a distinct cluster with HAdV-37p. In this cluster, f2 and f3 were located on the same branch. However, f4 was located on a different branch from f2 and f3 (Fig. 4).
When we compared the genome type and hexon/fiber sequence types, we found six pairs: hx2/f1, hx1/f1, hx1/f2, hx1/f3, hx3/f4, and hx4/f4. Except for the one D6 strain, isolated in 1990, the hexon and fiber sequences of each genome type belonged to a single hexon/fiber pair (Table 6).
Annual distribution of genome type and hexon/fiber pairing of HAdV-37. The predominant genome type changed from year to year. The predominant genome type was D1 in 1990 and 1991, but D7 in 1992. From 1993 to 1997, it switched from D6 to D8. After 1999, new genome types D10 and D11 emerged (Table 3). According to hexon/fiber pairing, six hexon/fiber pairs also emerged over 10 years. The predominant hexon/fiber types were hx1/f1 and hx2/f1 between 1990 and 1992 (Tables 3 and 6). The predominant type changed to hx1/f2 in 1993 and gradually switched to hx1/f3 between 1995 and 1997. After 1999, new hexon and fiber types emerged. In 1999, hx3/f4 was dominant, and in 2001, hx4 emerged while hx3/f4 and hx4/f4 were still detected. Analysis of the three epidemics of HAdV-37 in Sapporo (Fig. 1) shows that the fiber types were well separated from epidemic to epidemic. The first epidemic, in 1990 and 1991, corresponded to f1. The second, during 1994 to 1997, corresponded to f2 and f3, and the last epidemic after 1999 corresponded to f4.
DISCUSSION
In this study, we isolated eight genome types of HAdV-37 in Sapporo continually from 1990 to 2001 and confirmed two major findings. First, many more genome types were observed during our 12-year study than were observed in the previous reports (2, 13, 14, 21, 22, 26), and the genome types in our study appeared to be closely related to each other. Second, the hexon and fiber sequences changed in conjunction with alternations in the genome type, and significant transitions in genome types were observed for HAdV-37, as with other serotypes (1, 10, 15, 16, 20).
It has already been reported that the genetic variability of HAdV-37 is smaller than that of other adenovirus serotypes (2, 21). The restriction analysis of several HAdV-37 strains isolated between 1976 and 1983 in Europe, Australia, the United States, and Japan showed that HAdV-37 variants shared more than 90% of their restriction fragments (2, 17). In our study, the eight HAdV-37 variants shared more than 95% of their restriction fragments with those of HAdV-37p. Consequently, the genetic stability observed previously for HAdV-37 was confirmed here. However, no other reports have demonstrated such a large number of different genome types isolated in only one city. For further analysis of the genetic variability of the HAdV-37 genome, we sequenced partial hexon and full-length fiber genes of the eight genome types. We reported earlier on the phylogenetic analysis based on the partial hexon gene (29) and found this analysis to be useful for the classification of adenoviral isolates. In the present study, we analyzed hexon genes in conjunction with fiber genes, since serotype-specific antigens exist in fiber proteins also (32). Throughout our investigation, the hexon/fiber sequence types were well correlated with genome type and changed in conjunction with genome type alteration (Table 6). Consequently, we concluded that nucleotide sequence analysis of these two regions corresponded well to the genome type analysis and that this analysis was useful for the genetic classification of HAdV-37 strains also. Among the four clusters of 916-bp hexon gene fragments that were analyzed, we identified five point mutations. The homology ratio of hx2 to hx4 and hx1, the prototype hexon gene, was 99.5 to 99.9%. Among the fiber genes, there were three point mutation sites, and the homology ratio of the f2 to f4 genes and f1, the prototype fiber, was 99.8 to 99.9%. Among other serotypes that cause ocular infections (such as HAdV-4a and HAdV-19a), the homology rate of partial hexon genes as a percentage of the respective prototype strains was found to be 96.1 (6) or 96.9% (29). Consequently, the homology rates of HAdV-37p and the HAdV-37 variants identified in this study were higher than those of the other serotypes, and the genetic stability of HAdV-37 was confirmed by nucleotide sequence analysis.
As mentioned above, genome type transitions have been observed for many serotypes, and changes in genome types have been correlated with certain HAdV epidemics (1, 10, 16, 20). In this study, we observed that HAdV-37/D1, HAdV-37/D3, and HAdV-37/D6 to HAdV-37/D11 emerged chronologically. Consequently, we confirmed that the transition of genome types also occurs for HAdV-37 as it does for other serotypes. However, since there was little differentiation among restriction fragments from genome type to genome type identified in this study, it is difficult to examine in detail the genetic relationships between genome types by DNA restriction analysis alone. For a more detailed analysis between genome types, nucleotide sequence analysis was more useful. In the early 1990s, two hexon types, hx1 and hx2, were identified. After 1993, the hx2 type hexon disappeared, and hx1 dominated from 1994 to 1998. However, after 1999 hx1 disappeared, and two new hexon types, hx3 and hx4, emerged. Nucleotide transitions were observed even more clearly by the analysis of fiber gene sequences. The predominant fiber type shifted from f1 to f2, f3, and f4 in that order (Tables 3 and 6). One point mutation was detected in a comparison between f1 and f2. Fiber type f3 contained one more mutation than f2. It seems as though the f1 fiber mutated to f2 and that the f2 fiber mutated to f3. These mutations occurred between 1990 and 1997. The change in the hexon sequence in the same period was at only one site (hx1 and hx2). Thus, the strains isolated between 1990 and 1997—genome types D1, D3, and D6 to D9—were considered to be closely related. However, types D10 and D11, which were isolated after 1999, were not so closely related to old genome types by hexon and fiber sequences. Hexon sequences hx3 and hx4 formed a cluster different from that of hx1 and hx2, and f4 was located on a branch different from that of f2 and f3 (Fig. 3 and 4). It is possible that the D10 and D11 genome types were introduced recently from outside Sapporo. Amino acid mutations have occurred in the shaft domain of the fiber proteins in only f2, f3, and f4; this is notable because fiber protein is the means by which HAdV attaches to target cells (28). However, the biological consequences of these mutations are unknown, and it is possible that they have no influence on the virus-receptor interaction. Nevertheless, in this study different fiber sequences were detected from three epidemics of adenoviral keratoconjunctivitis in Sapporo. Further analysis is required to elucidate the relationships between these fiber protein sequences and the epidemics.
In conclusion, we investigated the genome types and nucleotide sequences of HAdV-37 strains isolated from patients with adenoviral keratoconjunctivitis in Sapporo. We identified eight genome types, including five new ones. These eight genome types showed clear transitions in the predominant genome type over time. All three epidemics of adenoviral keratoconjunctivitis in Sapporo between 1990 and 2001 were associated with certain genome types, and each contained specific fiber sequences. Comparative studies of fiber genes are expected to contribute to the understanding of the epidemiology of ocular adenoviruses.
ACKNOWLEDGMENTS
This study was supported in part by a grant from the Takeda Science Foundation, in part by a grant for Researches on Sensory and Communicative Disorders from the Ministry of Health, Labor and Welfare, Japan, and in part by grants-in-aid for Scientific Research from the Ministry of Education, Culture, Sports, Science, and Technology, Japan.
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