B R I E F R E P O R T
Secular trend of genome types of respiratory adenovirus type 3
during 1983–2005: a study from Taiwan
Yung-Cheng Lin•Kuei-Hsiang Lin •
Guan-Ming Ke•Min-Sheng Lee•Pei-Chin Lin• Hsin-Fu Liu• Li-Ching Hsu• Po-Liang Lu
Received: 8 August 2009 / Accepted: 30 November 2009 / Published online: 24 December 2009 Ó Springer-Verlag 2009
Abstract Genome type analysis of adenovirus type 3 (Ad3) in Taiwan identified four types (Ad3a, Ad3a2, Ad3a1, Ad3–7) during 1983–2005. Ad3a was the major type during 1983–1999, while Ad3a2 was the predomi-nant type from 2001 to 2005. Phylogenetic analysis of the hexon gene of 23 isolates revealed that most Ad3a2 and Ad3–7 isolates belonged to one cluster, and most Ad3a isolates to the other cluster. The clinical manifestations included respiratory tract infections, acute gastroenteritis, hand-foot-and-mouth disease, febrile convulsion and pharyngoconjunctival fever. In conclusion, Ad3a2 has replaced Ad3a as the most common genome type in Taiwan since 2001.
Introduction
Adenovirus is a clinically important pathogen, causing 5–10% of lower respiratory tract infections in infants and young children [20]. There are 51 known human serotypes, which have been classified into six species (A–F). Only one-third of the 51 serotypes can cause human diseases. Species B (Ad3, 7, 14, 16, 21, 34, 35), species C (Ad1, 2, 5, 6), and species E (Ad4) are responsible for acute respiratory diseases in infants, young children, and military recruits [11, 12, 16, 21, 36, 38, 39]. The serotypes fre-quently recovered from children with respiratory tract infections are Ad1, Ad2, Ad3, Ad5 and Ad7 [20]. Among them, Ad3 accounted for 13% of respiratory infection in a survey of respiratory infections from 1967 to 1976 and is one of the most common causative agents of acute
Y.-C. Lin and K.-H. Lin contributed equally to this work. Y.-C. Lin K.-H. Lin G.-M. Ke
Department of Clinical Laboratory,
Kaohsiung Medical University, Kaohsiung, Taiwan Y.-C. Lin H.-F. Liu
Institute of Bioscience and Biotechnology,
National Taiwan Ocean University, Keelung, Taiwan K.-H. Lin P.-L. Lu
Department of Laboratory Medicine, Kaohsiung Medical University Hospital,
Kaohsiung Medical University, Kaohsiung, Taiwan M.-S. Lee P.-C. Lin
Department of Pediatrics,
Kaohsiung Medical University Hospital,
Kaohsiung Medical University, Kaohsiung, Taiwan H.-F. Liu
Department of Medical Research,
Mackay Memorial Hospital, Tamshui, Taiwan
L.-C. Hsu
Department of Health, Center for Disease Control, Tainan, Taiwan
P.-L. Lu
School of Medicine,
Kaohsiung Medical University, Kaohsiung, Taiwan P.-L. Lu (&)
Department of Internal Medicine, Kaohsiung Medical University Hospital,
Kaohsiung Medical University, 100 Tzyou 1st Road, Kaohsiung, Taiwan
e-mail: [email protected]; [email protected] DOI 10.1007/s00705-009-0575-6
respiratory infections, pharyngoconjunctival fever (PCF) and conjunctivitis, with a worldwide distribution [2,3,14,
17,18,27,30,32,33,39].
At least 38 genome types of Ad3 have been identified worldwide. They include Ad3p (the prototype), Ad3p1– Ad3p3, Ad3a, Ad3a1–Ad3a18, Ad3b, Ad3b1, Ad3c–Ad3e, Ad3e1, Ad3e2, Ad3f–Ad3k, Ad3x and Ad3–7 [2–5,13–16,
23,29,38]. The nomenclature system was proposed by Li and Wadell [23] and subsequently modified by Li et al. [19,20,24] based on the restriction fragment length poly-morphism (RFLP) patterns. In Taiwan, adenoviruses are the second most common viral agent (4%), after RSV (7%) and followed by enterovirus (3%), in children hospitalized with respiratory infection [35]. With the exception of outbreaks due to Ad7 in 1999 and Ad4 in 2000 and 2001, Ad3 has existed as the most common serotype during the past two decades [25,26]. However, the molecular epidemiology of Ad3 in Taiwan has only been reported in a study from northern Taiwan [6]. Ad3 genome types varied with time and geographic areas and were associated with different disease severity [14,18,19,37]. To understand the preva-lence and chronological change in genome types of Ad3 in Taiwan, we did genetic analysis of isolates from each year using RFLP for viral DNA genome typing and analysis of the nucleotide and amino acid sequence of the full hexon gene. Clinical characteristics of Ad3-infected patients were also studied in correlation with the Ad3 genome types.
Materials and methods
Adenovirus isolated from nasopharyngeal and eye speci-mens of patients visiting Kaohsiung Medical University Hospital from 1983 to 2005 were included for serotyping. Viruses were grown in HEp-2 or A549 cells for 3–5 days and identified by immunofluorescent assay using an ade-novirus-specific monoclonal antibody (Chemicon, CA, USA). Virus stocks were prepared in HEp-2 cells with Eagle’s minimum essential medium supplemented with 2% fetal calf serum and antibiotics.
Adenovirus serotypes were identified by PCR-RFLP as described in a previous report [31]. Viral DNA used for PCR-RFLP was extracted using a blood purification kit (Amershan Pharmacia Biotech Inc., NJ, USA). The primer pair AdnU-S’ (50-TTCCCCATGGCNCACAACAC-30) and AdnU-A (50-GCCTCGATGACGCCGCGGTG-30) were used to amplify a 956-bp product from the hexon region. PCR products were subjected to digestion with three restriction enzymes, StyI, HaeIII and HinfI (Promega, Madison, WI, USA), according to the manufacturer’s instructions. The digested products were then electrophore-sed on a 3% agarose gel containing ethidium bromide (0.5 lg/ml).
Viral DNA for RFLP analysis was extracted according to a previously reported method [25]. Briefly, the infected cells were lysed with SDS (1%) and digested with pro-teinase K (50 lg/ml) at 65°C for 1 h. DNA was extracted twice with phenol–chloroform-isoamyl alcohol (25:24:1) followed by RNase (100 lg/ml) treatment at 65°C for 30 min. Isolates identified as Ad3 by PCR-RFLP were subjected to RFLP analysis for genotyping. Aliquots con-taining 1–2 lg of viral DNA were digested with 10–15 U of BamHI, HindIII, SmaI, XhoI, or BglI (Promega, Madison, WI, USA) according to the manufacturer’s instructions. After enzyme digestion, viral DNA was electrophoresed on a 1% agarose gel containing ethidium bromide (0.5 lg/ml) for 16 h at 50 V. The RFLP patterns were identified according to the patterns of genome types reported previously [23,26].
Four primers (AdHxF 50-GCCGAGGCTGAGTTGC TTTCA-30; AdHxR 50-CGCTGGAGCCGTTTCCGGA C-30; AdHxF-1, 50-AACGTCTGATAACTCTCATG-30; and AdHxR-1 50-CAGTTGCGAGATGGGATGGA-30) were used to amplify the full-length hexon gene sequence (2,835 bp). Products purified with QIAquick spin columns (Qiagen,Valencia, CA, USA) were used for sequencing.
For phylogenetic analysis, the hexon gene sequence data were analyzed using the DNASTAR Lasergene software (DNASTAR Inc., Madison, WI, USA). Phylogenetic analysis was conducted by the neighbor-joining method with MEGA 3 software (Molecular Evolutionary Genetics Analysis, Version 3.0) [22]. The robustness of the NJ tree was evaluated statistically by bootstrap analysis with 1,000 bootstrap samples. The demographics and the clinical characteristics of 65 patients for whom adequate informa-tion was available were analyzed.
Results
Ad3 accounted for 68 and 44% of adenovirus isolated during the periods of 1981–1989 and 1990–1998, respectively. Ad3 comprised of 36–81% of adenovirus isolated each year from 1999 to 2005. It was the most common serotype in the last two decades except in 1999 and 2001, when large outbreaks of Ad7 and Ad4, respectively, occurred.
Genome typing of Ad3 by RFLP
Ninety-two Ad3 isolates were randomly selected for gen-ome type analysis. The number of isolates for gengen-ome typing for each time period is in proportion to the total amount of Ad3 in that time period, with the exception of the unusually large number in 2005 due to an epidemic outbreak [29]. Genome typing of Ad3 by full-genome RFLP identified four genome types, Ad3a, Ad3a1, Ad3a2
and Ad3–7 during 1983–2005. Ad3a predominated during the period of 1983–1999 (53%, 14/26), while Ad3a2 pre-dominated in 2001–2005 (80%, 36/46) (P B 0.001) (Table1). Both Ad3a and Ad3a2 were found in similar proportions in 2000 (48 vs. 52%). Ad3–7 was found spo-radically in 1999 and 2003, while only one strain of Ad3a1 was found in 1983.
Comparison of nucleotide and amino acid sequences in the hexon region
Phylogenetic analysis showed that Ad3 isolates from 1983 to 2005 in our area grouped into two major clusters, which were separate from the prototype (GB strain, GenBank accession numbers X76549 and AY599834) and strains isolated from Korea. Cluster 1 consisted of most Ad3 isolates in 1983–1999, while Cluster 2 included most Ad3 isolates from 2001 to 2005 (Fig.1). Three Chinese strains belonged to cluster 1 and the USA strain belonged to cluster 2. Unique nucleotides were found in cluster 1 and cluster 2 at seven positions: 614(T-G), 1,249(A-G), 1,285(A-G), 1,316(C-A), 1,731(T-C), 2,061(T-C), and 2,067(T-C), which led to changes in four amino acid res-idues: V205G, N417H, T429A, and A439D.
Clinical features of patients
The most common age of disease occurrence was 4 years old, and 79% were less than 7 years old. None of the cases had a fatal outcome. The ratio of males to females was 1.4:1. Twenty-five of the 65 patients (38.5%) had acute febrile pharyngotonsillitis. Lower respiratory tract infec-tions such as acute bronchitis (29.2%, 19/65) and pneu-monia (21.5%, 14/65) accounted for half of the cases. There was a higher percentage of Ad3a2 than of Ad3a (95.1 vs. 79.1%) in respiratory tract infections. Complica-tions such as acute otitis media and acute sinusitis that made clinicians prescribe antimicrobial agents were found in 6 of 41 (14.6%) Ad3a2-infected patients, but not in any of the Ad3a cases. Acute gastroenteritis was found in 16.7% of Ad3a cases, but gastroenteritis did not occur in
Ad3a2-infected cases (p value = 0.016, Fisher’s exact test) (Table2).
Discussion
RFLP of the whole genome of adenovirus is a useful tool in molecular epidemiology [24]. A sequence library for ade-noviruses typing was established in 2004 based on a 956-bp
Table 1 Chronological change of Ad3 genome types during 1983– 2005
Genotype no. (%) Total 1983–1999 2000 2001–2005 Ad3a 14 (53.8) 10 (47.6) 5 (11.1) 29 (31.5) Ad3a1 1 (3.8) 0 0 1 (1.1) Ad3a2 5 (19.2) 11 (52.4) 36 (80) 52 (56.5) Ad3–7 6 (23.1) 0 4 (8.9) 10 (10.9) Total 26 21 45 92
Fig. 1 Phylogenetic analysis of nucleotide sequences of full-length hexon genes of Ad3 isolates. The phylogenetic tree was constructed by the neighbor-joining method and using bootstrap analysis (n = 1,000) to determine the best-fitting tree. Sequences available in GenBank include Korean isolates whose names start with KNIH, Chinese isolates (AY878716, DQ099432, DQ105654), a USA isolate (AY599836), the prototype GB strain and X76549
partial hexon sequence [34]. Nowadays, instead of the neutralization test, PCR-RFLP and sequence analysis of the partial hexon region are both important methods for epi-demiological studies of adenovirus. Our phylogenetic study on the whole hexon gene had importance because serotype-specific epitopes are contained in the gene [8]. Phyloge-netic analysis of the Ad3 hexon gene sequences among isolates from Taiwan revealed two clusters, and unique signatures of nucleotide and amino acid sequences were observed in different clusters of the hexon gene sequences. However, not every genome type uniquely belonged to a specific cluster by hexon gene analysis when comparing the hexon gene cluster result and the genome types (Fig.1). Most Ad3a2 and Ad3–7 isolates were located in cluster 2 except the two Ad3a2 isolates from earlier years (isolate 312-92-TW from 1992 and isolate 846-96-TW from 1996), which belonged to cluster 1. Comparison of full hexon gene sequence analysis and human adenovirus serotypes in previous studies has revealed a discrepancy in grouping results in serotype 16, 50 (species B) and 51 (species D) [10]. The correlation of genome type and the result of phylogenetic analysis of the hexon gene for Korea Ad3 isolates showed that isolates of specific genome types were clustered in the phylogenetic tree [7]. In contrast, there was non-correlation between the results of genome typing and phylogenetic analysis on the hexon gene in our results.
The fact that Ad3a isolates that did not belong to cluster 1 were isolated both in 2004 and 2005 suggested there were nucleotides changes occurring in the hexon gene in the Ad3a genome type in the later years of our study period. Coincidentally, there was a community-derived outbreak of Ad3 in Taiwan in 2004 and 2005 [6]. The outbreak might be related to the fact that the hexon gene contains hyper-variable regions, and the evolution of the adenovirus serotype is suggested to be driven by changes in the hypervariable regions of the hexon protein [9]. The chan-ges of hexon gene nucleotides of Ad3a and Ad3a2 over time necessitates further observation, since fatal acute respiratory disease may be associated with variant-type
adenoviruses, as exemplified by outbreaks of adenovirus serotype 14 in the USA in 2006–2007 [1]. Increasing numbers of cases of Ad3 have also been reported, with the identification of new genome types [20,28].
Although there are 38 genome types of Ad3 in the world, only a few genome types occur in a specific geo-graphic area. Chronological change in the genome type has been reported in specific geographic areas [20, 24]. Het-erogeneity of genome types in an epidemic of Ad3 has been observed before [20]. Regarding the Ad3 genome types in the East Asia area, Ad3a2 was dominant, with occasional isolates of Ad3a4, Ad3a5 and Ad3a6 during the period from 1962 to 1988 in China [24]. In Japan, Ad3a was the predominant genome type, with occasional isolates of Ad3a8 and Ad3c from 1983 to 1991 [17]. For con-junctivitis manifestation in Japan, the predominant role of Ad3g was taken over by Ad3f during 1983–1986 [15]. In Korea, Ad3a13, Ad3a14, Ad3a15, Ad3a16, Ad3a17 and Ad3a18 were identified as new variants of Ad3a during the period of 1990–2000, and the dominant genome types were Ad3a16 and Ad3a13. Ad3 genome types in Korea are more diverse than those observed in China, Japan and Taiwan [13]. In this study, four genome types (Ad3a, Ad3a1, Ad3a2, Ad3–7) were identified in Taiwan. Ad3a was dominant during 1983–1999 and Ad3a2 was dominant in 2001–2005. Although Taiwan is a neighbor of China, Japan, and Korea and there is frequent traffic communi-cation between these countries, different Ad3 genome type distributions were observed in the different countries.
Half of the Ad3a and Ad3a2 cases had a diagnosis of acute lower respiratory tract infection, such as pneumonia and bronchitis. While no gastroenteritis cases occurred among Ad3a2-infected patients, some Ad3a cases mani-fested mainly with acute gastroenteritis. Although Ad3 is not regarded as a gastroenteritis adenovirus like species F (Ad40 and Ad41), more than half of respiratory Ad3 infection cases may be accompanied by gastrointestinal discomfort symptoms [6]. Since some genome types may be associated with different disease presentations, as our results showed that clinical presentations of Ad3a and
Table 2 Clinical features of 65 Ad3-infected patients during 1992–2005
a Hand-foot-and-mouth disease
(2 cases) and febrile convulsion (1 case)
b One Ad3a and 2 Ad3a2 cases
were pharyngoconjunctival fever
Ad3a Ad3a2 Total
Number of cases (%) 24 (36.9) 41 (63.1) 65 (100) Age (years): median (range) 4 (0–9) 5 (0–63) 4 (0–63) Male/female (ratio) 13/11 (1.18) 25/16 (1.56) 38/27 (1.41) Clinical diagnosis [no.] (%)
Acute pharyngotonsillitisb(%) 8 (33.3) 17 (41.5) 25 (38.5) Acute bronchitis (%) 6 (25.0) 13 (31.7) 19 (29.2) Pneumonia (%) 5 (20.8) 13 (31.7) 14 (21.5) Acute gastroenteritis (%) 4 (16.7) 0 (0.0) 4 (6.2) Othera(%) 1 (4.2) 2 (4.9) 3 (4.6)
Ad3a2 differed, the correlation of genome types and clin-ical manifestations will require a larger number of cases for confirmation.
In conclusion, we observed the genome type change of Ad3 in Taiwan from 1983 to 2005 with difference in clinical manifestations. Ad3a and Ad3a2 were the pre-dominant genome types before and after 2000, respec-tively, with Ad3a1 and Ad3–7 being isolated occasionally.
Acknowledgments This work was supported by grants from the Center for Disease Control and Prevention of Taiwan (92-DC-1201 and 94-DC-003). We thank Li-Chiu Chou, Hui-Ju Su, Ju-Feng Wang, Liang-Yin Ke and Yi-Yin Lin for their technical assistance.
References
1. Centers for Disease Control and Prevention (2007) Acute respi-ratory disease associated with adenovirus serotype 14–four states, 2006–2007. MMWR Morb Mortal Wkly Rep 56:1181–1184 2. Adrian T, Best B, Hierholzer JC, Wigand R (1989) Molecular
epidemiology and restriction site mapping of adenovirus type 3 genome types. J Clin Microbiol 27:1329–1334
3. Adrian T, Wigand R, Knocke KW, Schafer G, Grundmann M (1989) Genome type analysis of adenoviruses: isolates from one year from the Hannover area. Arch Virol 105:89–101
4. Arens M, Dilworth V (1988) Remarkably homogeneous popula-tion of adenovirus type 3 and 7 genome types. J Clin Microbiol 26:1604–1608
5. Bailey AS, Richmond SJ (1986) Genetic heterogeneity of recent isolates of adenovirus types 3, 4, and 7. J Clin Microbiol 24:30–35 6. Chang SY, Lee CN, Lin PH, Huang HH, Chang LY, Ko W, Chang SF, Lee PI, Huang LM, Kao CL (2008) A community-derived outbreak of adenovirus type 3 in children in Taiwan between 2004 and 2005. J Med Virol 80:102–112
7. Choi EH, Kim HS, Park KH, Lee HJ (2006) Genetic heteroge-neity of the hexon gene of adenovirus type 3 over a 9-year period in Korea. J Med Virol 78:379–383
8. Crawford-Miksza L, Schnurr DP (1996) Analysis of 15 adeno-virus hexon proteins reveals the location and structure of seven hypervariable regions containing serotype-specific residues. J Virol 70:1836–1844
9. Crawford-Miksza LK, Schnurr DP (1996) Adenovirus serotype evolution is driven by illegitimate recombination in the hyper-variable regions of the hexon protein. Virology 224:357–367 10. Ebner K, Pinsker W, Lion T (2005) Comparative sequence
analysis of the hexon gene in the entire spectrum of human adenovirus serotypes: phylogenetic, taxonomic, and clinical implications. J Virol 79:12635–12642
11. Echavarria M, Sanchez JL, Kolavic-Gray SA, Polyak CS, Mitchell-Raymundo F, Innis BL, Vaughn D, Reynolds R, Binn LN (2003) Rapid detection of adenovirus in throat swab speci-mens by PCR during respiratory disease outbreaks among mili-tary recruits. J Clin Microbiol 41:810–812
12. Erdman DD, Xu W, Gerber SI, Gray GC, Schnurr D, Kajon AE, Anderson LJ (2002) Molecular epidemiology of adenovirus type 7 in the United States, 1966–2000. Emerg Infect Dis 8:269–277 13. Golovina GI, Zolotaryov FN, Yurlova TI (1991) Sensitive anal-ysis of genetic heterogeneity of adenovirus types 3 and 7 in the Soviet Union. J Clin Microbiol 29:2313–2321
14. Guo DF, Shibata R, Shinagawa M, Sato G, Aoki K, Sawada H (1988) Genomic comparison of adenovirus type 3 isolates from
patients with acute conjunctivitis in Japan, Australia, and the Philippines. Microbiol Immunol 32:833–842
15. Itakura S, Aoki K, Sawada H, Shinagawa M (1990) Analysis with restriction endonucleases recognizing 4- or 5-base-pair sequences of human adenovirus type 3 isolated from ocular diseases in Sapporo, Japan. J Clin Microbiol 28:2365–2369
16. Itakura S, Aoki K, Sawada H, Ishiguro N, Shinagawa M (1991) Changes in subgenome types of adenovirus type 4 isolated from patients with ocular disease between 1985 and 1989 in Sapporo, Japan. J Clin Microbiol 29:1740–1743
17. Itoh N, Tanaka K, Aoki K, Hinokuma R, Nakagawa H, Takeuchi S, Uchio E, Shiao S, Ohno S (1999) Four new genotypes of adenovirus type 3 isolated from patients with conjunctivitis in Japan. J Med Virol 59:73–77
18. Kajon AE, Murtagh P, Garcia Franco S, Freire MC, Weissenb-acher MC, Zorzopulos J (1990) A new genome type of adeno-virus 3 associated with severe lower acute respiratory infection in children. J Med Virol 30:73–76
19. Kajon AE, Mistchenko AS, Videla C, Hortal M, Wadell G, Avendano LF (1996) Molecular epidemiology of adenovirus acute lower respiratory infections of children in the south cone of South America (1991–1994). J Med Virol 48:151–156
20. Kim YJ, Hong JY, Lee HJ, Shin SH, Kim YK, Inada T, Hashido M, Piedra PA (2003) Genome type analysis of adenovirus types 3 and 7 isolated during successive outbreaks of lower respiratory tract infections in children. J Clin Microbiol 41:4594–4599 21. Kolavic-Gray SA, Binn LN, Sanchez JL, Cersovsky SB, Polyak
CS, Mitchell-Raymundo F, Asher LV, Vaughn DW, Feighner BH, Innis BL (2002) Large epidemic of adenovirus type 4 infection among military trainees: epidemiological, clinical, and laboratory studies. Clin Infect Dis 35:808–818
22. Kumar S, Tamura K, Nei M (2004) MEGA3: integrated software for molecular evolutionary genetics analysis and sequence alignment. Brief Bioinform 5:150–163
23. Li QG, Wadell G (1988) Comparison of 17 genome types of adenovirus type 3 identified among strains recovered from six continents. J Clin Microbiol 26:1009–1015
24. Li QG, Zheng QJ, Liu YH, Wadell G (1996) Molecular epide-miology of adenovirus types 3 and 7 isolated from children with pneumonia in Beijing. J Med Virol 49:170–177
25. Lin KH, Chow TY, Sheu MM, Huang WL, Chen CW (1986) A rapid and simple method for preparation of adenovirus DNA for restriction endonuclease cleavage studies. Gaoxiong Yi Xue Ke Xue Za Zhi 2:774–777
26. Lin KH, Lin YC, Chen HL, Ke GM, Chiang CJ, Hwang KP, Chu PY, Lin JH, Liu DP, Chen HY (2004) A two decade survey of respiratory adenovirus in Taiwan: the reemergence of adenovirus types 7 and 4. J Med Virol 73:274–279
27. Mizuta K, Suzuki H, Ina Y, Yazaki N, Sakamoto M, Katsushima N, Numazaki Y (1994) Six-year longitudinal analysis of adeno-virus type 3 genome types isolated in Yamagata, Japan. J Med Virol 42:198–202
28. Noda M, Yoshida T, Sakaguchi T, Ikeda Y, Yamaoka K, Ogino T (2002) Molecular and epidemiological analyses of human ade-novirus type 7 strains isolated from the 1995 nationwide outbreak in Japan. J Clin Microbiol 40:140–145
29. O’Donnell B, Bell E, Payne SB, Mautner V, Desselberger U (1986) Genome analysis of species 3 adenoviruses isolated during summer outbreaks of conjunctivitis and pharyngoconjunctival fever in the Glasgow and London areas in 1981. J Med Virol 18:213–227
30. Rowe WP, Huebner RJ, Gilmore LK, Parrott RH, Ward TG (1953) Isolation of a cytopathogenic agent from human adenoids undergoing spontaneous degeneration in tissue culture. Proc Soc Exp Biol Med 84:570–573
31. Saitoh-Inagawa W, Oshima A, Aoki K, Itoh N, Isobe K, Uchio E, Ohno S, Nakajima H, Hata K, Ishiko H (1996) Rapid diagnosis of adenoviral conjunctivitis by PCR and restriction fragment length polymorphism analysis. J Clin Microbiol 34:2113–2116 32. Schmitz H, Wigand R, Heinrich W (1983) Worldwide
epidemiol-ogy of human adenovirus infections. Am J Epidemiol 117:455–466 33. Shiao S, Aoki K, Isobe K, Tsuzuki WL, Itoh N, Toba K, Kobayashi N, Noguchi Y, Ohno S (1996) Genome analysis of adenovirus type 3 isolated in Japan. J Clin Microbiol 34:413–416 34. Shimada Y, Ariga T, Tagawa Y, Aoki K, Ohno S, Ishiko H (2004) Molecular diagnosis of human adenoviruses d and e by a phylogeny-based classification method using a partial hexon sequence. J Clin Microbiol 42:1577–1584
35. Tsai HP, Kuo PH, Liu CC, Wang JR (2001) Respiratory viral infections among pediatric inpatients and outpatients in Taiwan from 1997 to 1999. J Clin Microbiol 39:111–118
36. Videla C, Carballal G, Kajon A (1999) Genomic analysis of adenovirus isolated from Argentinian children with acute lower respiratory infections. J Clin Virol 14:67–71
37. Wadell G, de Jong JC (1980) Restriction endonucleases in identification of a genome type of adenovirus 19 associated with keratoconjunctivitis. Infect Immun 27:292–296
38. Wadell G (1984) Molecular epidemiology of human adenovi-ruses. Curr Top Microbiol Immunol 110:191–220
39. Wadell G, Cooney MK, da Costa Linhares A, de Silva L, Kennett ML, Kono R, Gui-Fang R, Lindman K, Nascimento JP, Schoub BD et al (1985) Molecular epidemiology of adenoviruses: global distribution of adenovirus 7 genome types. J Clin Microbiol 21:403–408
