Eiko Suzuki

Molecular epidemiology of adenovirus infection among pediatric population with diarrhea in Asia

A total of 3,577 fecal specimens from infants and children with acute gastroenteritis in Japan, Korea and Vietnam during 1998 and 2001 were tested for adenovirus by the ELISA method. Of these, adenovirus was detected in 158 (4.4%). The detection rate of adenovirus was highest in Korea (8.7%, 20/231) followed by 5.0% (100/1,991) in Japan and 2.8% (38/1,355) in Vietnam. All adenoviruses were further serotyped by PCR‐RFLP. The diversity of adenovirus serotypes including Ad2, 3, 5, 8, 31, 40 and 41 was demonstrated. Worth of note was a decrease in the rate of isolation of Ad40 (7.6%, 12/158) and a concomitant increase of Ad41 (63.9%, 101/158) to become the predominant serotype. Another interesting feature of the study was the presence of Ad3 (4.0% and 30%) and Ad8 (8.0% and 20%) in Japan and Korea, respectively, which is mainly associated with keratoconjunctivitis worldwide. Our result underscored the importance of adenovirus in association with acute gastroenteritis in Asian countries.

Characterizations of Adenovirus Type 41 Isolates from Children with Acute Gastroenteritis in Japan, Vietnam, and Korea

ABSTRACT Genetic and antigenic characterizations of 70 strains of adenovirus type 41 (Ad41), isolated between 1998 and 2001 from children in Japan, Vietnam, and Korea, were done by DNA restriction enzyme (RE) analysis, sequencing analysis, and monoclonal antibody (MAb)-based enzyme-linked immunosorbent assay (ELISA). Eight genome types were observed in the present study, among which D25, D26, D27, and D28 were novel genome types. These eight genome types were divided into two genome-type clusters (GTCs) based on phylogenetic analysis of the hypervariable regions (HVRs) of the hexon. GTC1 includes D1, D25, D26, D27, and D28, and the GTC2 contains D4, D12, and D22. The amino acid homologies among the members within a GTC were 97 to 100%, whereas between the members of different GTCs the homologies were 92 to 94%. The specificity of the GTC classification was confirmed by ELISA with MAb 1F, which was selected by the Ad41 prototype Tak strain. It was found that only the isolates of GTC1 but not of GTC2 reacted with MAb 1F. These results suggest that Ad41 isolates from the three countries should be classified into two subtypes. The accumulation of amino acid mutations located in HVRs of hexon are indicative for the classification of Ad41 subtype.

Isolation and a preliminary characterization of temperature-sensitive mutants of adenovirus 12.

Eighty-eight temperature-sensitive ( ts ) mutants of adenovirus 12, which did not replicate at 38° but did replicate well at 31° were isolated. Thirty-four of these mutants were selected and classified into 13 groups (groups A to M) by complementation tests. Analyses of the defects in viral replication at the nonpermissive temperature (38°) revealed that mutants in 4 groups (groups A, B, C, and D) were defective in the production of hexon, fiber, and penton base, mutants in group E were defective in the production of both fiber and penton base, mutants in group F were defective in the production of both hexon and penton base, a mutant in group G was defective in the production of both hexon and fiber, mutants in group H were defective in the production of only hexon, mutants in group I were defective in the production of only fiber, mutants in group J were defective in the production of only penton base and mutants in 3 groups (groups K, L, and M) were not defective in the synthesis of hexon, fiber, and penton base. These defectiveness were detected by serological reaction and polyacrylamide gel electrophoresis. Immunofluorescent examinations of cells infected with these mutants as well as the examination of heat stability of virions produced at the permissive temperature showed differences among these groups.

Genetic characterisation of adenovirus type 8 isolated in Hiroshima city over a 15 year period

Aims: To investigate the genetic differences among the strains of adenovirus type 8 (Ad8) circulating in Hiroshima city, Japan, and to study their circulation pattern. Methods: One hundred and twenty nine strains of adenovirus type 8 (Ad8) were isolated in Hiroshima City over a 15 year period (1983–97) from patients with keratoconjunctivitis, and analysed with six restriction enzymes—BamHI, HindIII, PstI, SacI, SalI, and SmaI—to investigate possible relations among the isolates and their genetic variability. Seven hypervariable regions of the hexon gene that carry the type specific epitope were also sequenced to investigate the variation among the genome types. Results: Restriction endonuclease analyses yielded three known genome types (Ad8A, 13 samples; Ad8B, seven samples; and Ad8E, 35 samples) and a novel genome type (Ad8I, 74 samples). Ad8A, Ad8B, and Ad8E were closely related, with 96% homology, whereas Ad8I had only 71% homology. Ad8A, Ad8B, and Ad8E shared 91.8% and 96.4% homology with regard to their amino acid and nucleotide sequences, respectively, with the isolate 1127 (accession no X74663). However, when compared with Ad8A, Ad8B, Ad8E, and isolate 1127, Ad8I shared only 62.7% and 69.9% homology with regard to amino acid and nucleotide sequences, respectively. Ad8A, Ad8B, and Ad8E had a unique 31 amino acid deletion in the hypervariable region 1 of the hexon gene, whereas Ad8I had a 33 residue deletion. The Ad8E strain that circulated from 1984 to 1995 was stable among the study population. Ad8I was isolated from an outbreak of epidemic keratoconjunctivitis in 1995 and was also isolated from sporadic cases until 1997. Conclusions: These results confirmed that genetic variability occurs in Ad8 in the microenvironment and revealed the emergence of a new genome type (Ad8I).

Serological and genetic characterisation of a unique strain of adenovirus involved in an outbreak of epidemic keratoconjunctivitis

Aims: To characterise a novel strain of adenovirus (Ad) type Ad8 (genome type Ad8I) involved in an epidemic keratoconjunctivitis (EKC) outbreak in Hiroshima city using serological testing and sequence analysis of the fibre and hexon gene. Methods: A neutralisation test (NT) was performed in microtitre plates containing a confluent monolayer of A549 cells using 100 tissue culture infectious doses of virus and type specific antisera. The haemagglutination inhibition test was also carried out in microtitre plates with rat erythrocytes using four haemagglutination units of virus and twofold dilutions of serum. The fibre gene was sequenced by generating overlapping polymerase chain reaction products or by direct sequencing of genomic DNA. Primer selection was based on alignment of the fibre genes of human adenovirus serotypes Ad8, Ad19, Ad37, Ad9, and Ad15 available from Gene Bank. Results: The virus strain was specifically neutralised by anti-Ad8 antibodies, although there was a major crossreaction with anti-Ad9 antibodies. Haemagglutination was equally inhibited by anti-Ad8 and anti-Ad9 antibodies. The predicted amino acid sequences of the hypervariable regions (HVRs) of the Ad8I hexon gene showed higher homology with Ad9 (83.3%) than with Ad8 (62.0%). However, the Ad8I fibre knob was more homologous to Ad8 (94.4%) than to Ad9 (91.6%). Conclusions: Ad8I is a unique strain of adenovirus because of its lower genomic homology with Ad8, major crossreactivity with Ad9 in NT, and mixed genetic organisation of HVRs of the hexon gene. These factors may have enabled the virus to circumvent acquired immunity, resulting in the outbreak.

A temperature-sensitive mutant of adenovirus 31, defective in viral deoxyribonucleic acid replication

Abstract A temperature-sensitive mutant ( ts 13) of adenovirus 31 was isolated. The analysis of the replication of the mutant in human embryonic kidney cells at the nonpermissive temperature (39.5°) revealed that the T antigen, deoxyribonucleic acid (DNA) polymerase, and thymidine kinase were induced, but viral DNA and capsid proteins were not synthesized. When the incubation temperature was shifted-down from 39.5° to 33.5° at 16 or 20 hr post infection, viral DNA synthesis was observed normally but shift-down at later time resulted in decrease of viral DNA synthesis. No DNA synthesis was observed when cycloheximide was added at the time of shift-down. When temperature was shifted-up during viral DNA replication, the rate of DNA synthesis was first accelerated for a short period, then followed by rapid decrease. The mutant produced tumors in hamsters and induced cellular DNA synthesis in nongrowing hamster cells at the nonpermissive temperature.

Characterisation of hexon and fibre genes of a novel strain of adenovirus involved in epidemic keratoconjunctivitis

Aims: To characterise a novel strain (M86) of adenovirus (Ad) involved in epidemic keratoconjunctivitis (EKC). Methods/Results: The virus strain was neutralised by antisera to both Ad35 and Ad11. Restriction endonuclease analysis of genomic DNA showed 98% and 88% homology with Ad11 and Ad35, respectively. The deduced amino acid sequence of the hypervariable regions of (HVRs) of the hexon gene showed a higher homology with Ad35 (94.4%) than with Ad11 (83.7%). However, it was 100% homologous to Ad35 in HVRs 1, 2, 3, and 6 and to Ad11 in HVRs 4 and 6. In the fibre knob, the isolate was more homologous to Ad11 (99.4%) than to Ad35 (29.1%). Conclusion: This novel strain of adenovirus showed similarities with both Ad11 and Ad35. The isolation of a novel strain like Ad35+11 is important because of its association with EKC.

Heterogeneity of the fibre sequence in subgenus C adenoviruses

Aims: To determine the nucleotide sequences of adenovirus (Ad) types 1 and 6 fibre genes; to clarify the molecular basis of the distinct haemagglutination properties of subgenus C Ads and their phylogenetic relations. Methods: Human Ad1 and Ad6 fibre genes were sequenced from genomic DNA by direct sequencing. Primer selection was based on alignment of the fibre gene of human Ad serotypes Ad2 and Ad5. Fibre based subgenus C specific polymerase chain reaction (PCR) was performed to check for deletions in field isolates of Ad6, as revealed by sequence analysis of the Ad6 prototype. A phylogenetic tree was constructed from the predicted amino acid (AA) sequences of the fibre gene of important Ads. Results: Ad1 and Ad6 comprise 1746 and 1584 nucleotides, encoding 582 and 528 AA, respectively. Ad6 showed deletions in motifs 15–17 (51 AA) of the shaft when compared with Ad1, Ad2, and Ad5. Subgenus C specific PCR with both prototype and field isolates also showed deletions in Ad6. In the shaft and knob, AA homology was 58.82–72.91% and 68.89–74.59%, respectively. The tail was 100% conserved. Phylogenetically, Ad1 and Ad6, including Ad2 and Ad5, formed a subgenus specific cluster, like other serotypes. Conclusions: The fibre gene (including the knob region) of subgenus C Ads is heterogeneous, providing the molecular basis for lack of crossreactivity in the haemagglutination inhibition test. This heterogeneity could be helpful in fibre based genotyping of subgenus C field isolates. Phylogeny might be useful for subgenus specific identification of important field strains.