We determined the complete genome sequence of epidemic keratoconjunctivitis (EKC)-related human adenoviruses (HAdVs). We analysed a total of 12 HAdV strains; three prototype strains and two HAdV-8, three HAdV-19 and three HAdV-37 clinical isolates from EKC patients in Japan, and one novel serotype of HAdV. Genome organization of these serotypes was identical to those of the recently determined HAdV-19 and HAdV-37. The identities of the whole genome were over 99 % among strains from the same serotype, except for HAdV-19p, which is not associated with conjunctivitis, resulting in the formation of a distinct cluster in the phylogenetic analysis. The penton, loop 1 and loop 2 of hexon, early region 3 (E3) and fiber were hypervariable regions between serotypes. Results suggest that the HAdV-19 clinical strain is a recombinant of HAdV-19p-like and HAdV-37-like strains, and that the acquisition of the penton, E3 or fiber may be related to ocular tropism.Adenoviruses are nonenveloped, double-stranded DNA viruses with icosahedral capsids (Swenson et al., 2003). Human adenoviruses (HAdVs) belong to the genus Mastadenovirus of the family Adenoviridae and are classified into six species, A to F (HAdV-A to HAdV-F) (Benkö et al., 2000;Wold & Horwitz, 2007). Adenoviral conjunctivitis is mainly caused by HAdV-3 (in HAdV-B), HAdV-4 (in HAdV-E), and HAdV-8, , with the three HAdV-D serotypes being known to cause epidemic keratoconjunctivitis (EKC). HAdV-8 is the original causative agent of EKC and remains the predominant HAdV serotype isolated in association with EKC in many countries (Ishii et al., 1987;Chang et al., 2001;Vainio et al., 2001;Aoki & Tagawa, 2002; Jin et al., 2006). In Japan, although HAdV-8 and HAdV-19 have been described, a novel serotype of HAdV recently isolated from EKC patients and HAdV-37 are the predominant causative serotype of EKC in Japan (Higuchi et al., 1987;Aoki & Tagawa, 2002;Ishiko et al., 2008;Kaneko et al., 2008).Nucleotide polymorphisms in HAdV strains isolated from EKC patients can be classified into discrete genotypes within a specific HAdV serotype on the basis of their restriction endonuclease cleavage pattern (Wadell et al., 1980;Adrian et al., 1986). It has been speculated that the appearance of new genotypes might contribute to the incidence of outbreaks of each serotype (Aoki & Tagawa, 2002;Ariga et al., 2005). DNA sequence analysis has allowed us to appreciate the molecular evolution of HAdV in greater detail, and revealed that the penton, hexon and fiber genes were the most variable among the different serotypes (Pring-Akerblom & Adrian, 1995;Arnberg et al., 1997;Ebner et al., 2005;Madisch et al., 2005Madisch et al., , 2007 MiuraOchiai et al., 2007). The complete genome sequences of 24 HAdV serotypes 2, 3, 4, 5, 7, 9, 11, 12, 14, 16, 17, 19, 21, 26, 34, 35, 37, 40, 41, 46, 48, 49 and 50) have now been determined.In this study, we describe the complete genome sequences of the prototype strains HAdV-8p, HAdV-19p and HAdV37p together with a novel HAdV serotype and eight clinical

Human adenoviruses (HAdVs) are the major causes of a variety of acute illnesses. Virus isolation and neutralization tests are usually done to identify the causative virus, but these tests are labor-intensive and time-consuming, and standardized antisera are in limited supply. This study investigated a rapid and reliable method of virus identification based on PCR and phylogenetic analysis. The phylogenetic tree constructed by neighbor joining on the basis of the newly determined partial hexon sequences from 33 prototypes of HAdV-D and -E, along with 11 available prototypes of HAdV-A to -C and -F from GenBank, allowed HAdVs to be grouped into six distinct clusters. These clusters correspond closely to the six newly designated species, HAdV-A to -F. The partial hexon sequences of 57 isolates from patients with acute conjunctivitis obtained over 20 years plus those of 44 prototype strains were analyzed. Each isolate formed a monophyletic cluster along with its respective prototype strain, allowing serotype identification. Partial-hexon-based classification appears to be an effective tool for studying the molecular epidemiology of HAdVs.

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.

Human adenovirus type 4 is one of the major serotypes isolated from patients with adenoviral conjunctivitis. In 2001 we encountered nosocomial infections with epidemic conjunctivitis in the ophthalmology ward of one hospital in Sapporo, which is in the northern part of Japan. Adenoviruses were isolated from the patients with this nosocomial infection and identified as adenovirus type 4 (AdV-4) by a neutralization test with serotypespecific antiserum. When the cleavage patterns of the isolates were compared with the full viral genome with BamHI and SmaI, the cleavage patterns of the isolates were shown to be different from those of AdV-4p and other previously known AdV-4 variants. The nucleotide sequences of the fiber gene of the isolates showed the highest homologies (94.3%) with AdV-4 among the nucleotide sequences available from GenBank and formed a monophyletic cluster along with the prototype strain of AdV-4. The isolates, however, were located in a different lineage from those of AdV-4p and the AdV-4 variant from the sporadic infections. We conclude that the nosocomial infection that appeared in 2001 was caused by a new genome type of AdV-4, which was designated AdV-4c.