The complete sequence of the hexon (2,805 bp, 934 amino acids [aa]), E3 peptides (4,387 bp), the fiber gene (1,175 bp, 325 aa), and E4 ORF 6/7 peptides was successfully determined for 12 strains of Ad7.

We identified previously unrecognized variations in the fiber gene and E4 ORF 6/7 peptides among the various genome types of Ad7. In particular, multiple amino acid changes at E4 ORF 6/7 peptides showed genome type–specific differences between Ad7d and a novel genome type Ad7l. However, no genetic divergence was identified in the hexon gene or in E3 peptides among the 12 Korean strains of Ad7. This result indicates that the nucleotide structures of the fiber gene and of E4 ORF 6/7 peptides might have contributed to the genetic heterogeneity during the Ad7 epidemics in Korea, whereas those of the hexon gene and E3 were highly conserved.

So far, the fiber gene and E4 have rarely been addressed with regard to the evolution and molecular epidemiology of Ad7 strains (17). Restriction analysis of genomic DNA has been the most popular method of describing the molecular epidemiology of the adenoviruses (18). However, little is known about sequence variations with regard to the genome type within an epidemic or between the epidemics. Previous observations indicate that sequence variations of the hexon gene and of the E3 ORF encoding 14.9-kDa protein may contribute to genetic heterogeneity and the evolutionary process of Ad7 strains (6,14,19,20).

Alignment of the amino acid sequences of the hexon gene demonstrated 2 genetic clusters, GTC1 (Ad7p and Ad7p1) and GTC2 (Ad7a, Ad7b, Ad7c, Ad7d, Ad7d2, Ad7g, and Ad7h), based on variations in the hypervariable regions (14). Variations between 2 clusters have been observed in variable region 1 because of 3-aa length differences and in the variable region 2 because of a substitution from 440 Leu to Gln, the latter of which dramatically affected the hydropathic character of this region. Moreover, mutations of hexon of Ad could play an important role in new outbreaks of adenoviral infection (14). Compared to the Chinese Ad7d strains of the early 1980s, 1 aa substitution from Cys (or Tyr) to Phe at position 100 was consistently observed in all Korean isolates among 8 mutation sites. However, the Ad7d and Ad7l Korean strains shared the same hexon gene sequence as Japanese Ad7d and Ad7d2 in 1992 and 1995. Phylogenetic tree analysis based on the hexon sequences suggested that the Ad7 isolates from Korea and Japan cluster into the same lineage and that this cluster is distinct from those of Chinese Ad7d and other strains. Therefore, the Korean Ad7d epidemic that began in 1995 may have been caused by the introduction of isolates that were prevalent in Japan from 1987 to 1992.

Fiber is a major constituent of adenovirus outer capsid (21). Fiber protein consists of a trimeric projection terminated by a knob (head) (21,22), and it plays a crucial role in adenoviral infection by allowing, possibly by direct interaction, the virus to attach to specific receptors on the host cell surface (23). In addition, variability in this region is expected to account for the observed serologic difference between serotype 3 and 7 fibers (24). Two variation sites observed in Ad7 strains are located at the knob region of the fiber gene near its carboxy-terminal end. All Korean Ad7 strains showed a substitution of Ala to Val at codon 112 with or without a change of Arg to Lys at codon 280, unlike the Japanese strains. Therefore, the Korean Ad7d strains are thought to be closely related to the Japanese strains with changes in the receptor domain of the fiber gene. Changes in the receptor-binding domain could influence the interaction between fiber and the host cell and possibly influence genetic heterogeneity in different geographic locations.

The E4 region of human adenoviruses encodes a set of proteins that can regulate early gene expression for viral RNA export and stabilization (21,25). The functions of E4 ORF 6/7 peptides of Ad7 are unknown, but the corresponding protein of adenovirus type 5 has the ability to induce the binding of cellular transcription factor E2F to the viral E2a promoter region (26). E2F induced by ORF 6/7 peptides might facilitate adenoviral infection under more stringent environments, such as in the absence of the E1A gene products (27,28). Furthermore, the 33.2-kDa ORF 6 protein is known to promote cell cycle–independent adenovirus growth (29).

Nucleotide changes specific to a novel genome type Ad7l were observed at 18 sites in E4 ORF 6/7 peptides. These substitutions have not been previously reported in published E4 sequences of Ad7 isolates from other parts of the world. Although available sequences are limited, a phylogenetic tree based on the sequences of fiber and E4 ORF 6/7 peptides showed evidence that genome type Ad7l has experienced substantially more changes than Ad7d strains. This finding strongly suggests that the E4 region can be important to molecular diversity and evolution of the Ad7 strains. Interplay between the host cell and the E4 of Ad7 may contribute to the observed distinctive features of genome types.

A total of 270 adenoviruses were identified from childhood pneumonia patients in our children's hospital during 10 years, 1990–2000. Of these 270 strains, Ad7d was not isolated until 1995, when explosive outbreaks occurred. Although we do not know the level of herd immunity to Ad7 at the beginning of epidemics, Ad7 had rarely circulated in Korea before the outbreak described in this study. Therefore, we assume that low immunity to Ad7 was critical to causing a large outbreak of Ad7. However, Noda et al. also reported that the mutation in the E3 of Ad7d2 strains could contribute to rapid spread during nationwide outbreaks in Japan (6).

As noted earlier, the nationwide outbreak of Ad7d in Korea in 1995 may have started with the introduction of the Japanese Ad7d strains, likely several years before 1995. To achieve a large outbreak, new Ad7d strains presumably required time to acquire greater access to hosts or the increased virulence acquired by fiber amino acid changes. However, a strain of novel genome type Ad7l was first detected in 1996, a year after the start of the Ad7d nationwide epidemic, spread rapidly during subsequent years, and then became the predominant genome type from 1998 to 2000. As reflected by their capacity to displace Ad7d, Ad7l might be more efficient than Ad7d for the dissemination in nonimmune hosts when a certain level of immunity was maintained. Whether the variations influence viral capacity for virulence and transmission during the epidemics is not currently understood; however, data shown in this study suggest that Ad7l was rapidly spread by multiple amino acid changes at fiber and E4 ORF 6/7 peptides during Ad7 outbreaks in Korea. This process might have been a consequence of viral adaptive strategies that allowed the virus to spread throughout Korea before the population had developed immunity.

In conclusion, the results presented here emphasize that fiber and E4 ORF 6/7 peptides may have roles in the evolutionary process and pathogenesis of Ad7 in Korea. The emergence of new genome types after the disappearance of a previously predominant type may be the result of type-specific host immune response or type-specific virulence, perhaps mediated by amino acid variations in fiber or E4. However, the mechanisms underlying viral adaptive processes and interactions between virus and host have to be established by future study to allow us to more effectively counteract highly virulent genome types.