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To understand the molecular basis of observed regional shifts in the genome types of adenovirus type 7 (Ad7) isolated in Korea during nationwide outbreaks from 1995 to 2000, the genetic variabilities of Ad7d and Ad7l were studied by sequence analysis of hexon, fiber, E3, and E4 open reading frame (ORF) 6/7 peptides. One amino acid change in the receptor-binding domain of fiber and 6 amino acid variations in E4 ORF 6/7 were identified between 2 genome types, while no variations were found in hexon and E3. Phylogenetic trees based on hexon, fiber, and E4 suggested that the Ad7 epidemic was probably caused by the introduction of the Japanese Ad7d strains. Our data also provide evidence that the rapid divergence of Ad7d to a novel genome type Ad7l could have been due to viral strategies involving multiple sequence changes in E4. This result suggests fiber and E4 ORF 6/7 peptides participate in the evolution of Ad7.

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.

Adenovirus is an important cause of respiratory infections in infants and children. Fifty-one serotypes have been identified, and adenovirus type 3 (Ad3) and Ad7 have often been associated with outbreaks of severe respiratory tract infections. Each serotype can be further divided into genome types based on the patterns of digestion of their DNAs with restriction enzymes. DNA restriction analysis was performed with 56 strains of Ad3 and 98 strains of Ad7 by using 12 restriction enzymes recognizing 6 bp ( Bam HI, Bcl I, Bgl I, Bgl II, Bst EII, Eco RI, Hin dIII, Hpa I, Sal I, Sma I, Xba I, and Xho I). The virus strains were isolated during outbreaks of lower respiratory tract infections in children during an 11-year period from 1990 to 2000 in Seoul, Korea. Among the Ad3 strains, seven genome types were identified; Ad3a and six novel types (Ad3a13, Ad3a14, Ad3a15, Ad3a16, Ad3a17, and Ad3a18). Multiple genome types cocirculated during outbreaks, and some of these were isolated during the 11-year observation period, while others were restricted to particular outbreaks. For Ad7, two genome types, Ad7d and Ad7l, the latter of which is a novel genome type, were identified. A shift in genome types occurred from Ad7d to Ad7l during successive outbreaks. Mortality was 3.6% among children with Ad3 infections and 18% among children infected with either of the Ad7 genome types. In conclusion, the data confirm that Ad3 genome types are more diverse than those of Ad7 and suggest that shifts of genome types may occur during successive outbreaks of Ad3 and Ad7.

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).

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.

Eye infections caused by adenovirus (Ad) often result in nosocomial infections and community epidemics with significant rates of morbidity. No antiviral agent effective against Ad is yet available for clinical use. Therefore, early diagnosis is still the mainstay for patient management and the prevention of epidemics. A multiplex PCR assay based on amplification of a combination of the fiber and hexon genes which can identify the six important oculopathogenic serotypes of Ads (Ad serotype 3[Ad3], Ad4, Ad7, Ad8, Ad19, and Ad37) in a single-tube amplification reaction was developed. Ad serotypes could be distinguished by the different amplicon sizes. The assay correctly identified prototype strains as well as isolates in clinical specimens. In comparison with a previously described PCR-restriction fragment polymorphism method, our assay gave unequivocal results for clinical specimens. Our multiplex PCR has the potential to serve as a rapid and cost-effective tool for the typing of important ocular Ads.

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.

Subgenus C human adenoviruses, which include serotypes 1, 2, 5, and 6, are often associated with respiratory illness, ocular infections, gastroenteritis, and systemic infection among immunocompromised patients. To address the problems associated with the conventional typing methods, we developed a fiber-based multiplex PCR assay for simple and specific identification of adenovirus type 1, 2, 5, and 6 field isolates. To design type-specific primers, adenovirus type 1 and 6 fiber genes were sequenced. The assay correctly identified prototype strains of adenovirus serotypes 1, 2, 5, 6, as well as 21 previously typed adenovirus field isolates. Mixing two different prototype DNAs produced two amplicons of different lengths, thus clearly distinguishing the prototypes. The results correlated 100% with serological tests and 95% with the previously described PCR-restriction fragment length polymorphism method. The detection of dual infection is an added benefit of the assay. No nonspecific amplification was detected with other adenovirus serotypes or with nonadenoviral DNA. Our fiber-based multiplex PCR assay will provide a convenient tool for type-specific identification of subgenus C adenovirus isolates in various clinical situations and in epidemiological investigations and is a better alternative than the hexon-based assay.

Cell-mediated immunity (CMI) to mouse adenovirus (M-Ad) infection was studied by macrophage migration inhibition test (MMI) as one of in vitro correlates of CMI. Both direct and indirect tests showed clearly that migration of packed peritoneal exudate cells (PEC) (immune mouse or nonimmune guinea pig) was remarkably inhibited; MIF was produced by interactions between immune PEC and infected cell extracts and between immune spleen cells and infected cells or their extracts. The antigen(s) responsible for the above MMI was demonstrated in 6- to 12-hour infected ME cells, and FUdR-treated infected ME cells. Since under these conditions there is S antigen(s) synthesis but not capsid antigen synthesis, the antigen(s) concerned must be an S antigen(s). T cells sensitized to infected cells were shown to be required to induce MMI. The MMI is specific for M-Ad, since no cross MMI was observed between M-Ad and SV40 systems. Time course study of the development of CMI to M-Ad by MMI tests showed that CMI became detectable 4 days post-infection (pi), reached its peak level about 10 days pi, and faded away rapidly in about 10 days thereafter.

The topological relationship on the mouse adenovirus (M-Ad)-infected cell surface between virus-induced specific cell surface(s) antigens and serologically defined major histocompatibility antigens (H-2) was analyzed by the cap formation technique. Rhodamine-isothiocyanate (RITC)-labeled anti-S serum failed to stain the surface of virus-infected lymphoid cells which were pretreated with anti-H-2 serum and fluorescein isothiocyanate (FITC)-labeled anti-mouse immunoglobulin serum (anti-M-Ig) to cap the appropriate H-2 antigens. Conversely, the capping of the S antigens by pretreatment with anti-S followed by FITC anti-M-Ig serum induced cocapping of H-2 antigens. The β2 microglobulins (β2m) were also shown to be cocapped with S antigens by anti-β2m or by anti-S serum. The S antigens, however, did not cocap with mouse-immunoglobulins or Thyl. 2 antigens on virus-infected B or T lymphocytes, respectively. To further elucidate the molecular relationship between S and H-2 antigens, radio-iodinated virus-infected cells were solubilized with Nonidet P40 (NP40) and S antigens were precipitated with anti-S serum. When the precipitates were analysed with sodium dodecyl sulfate Polyacrylamide gel electrophoresis, two major peaks were seen at positions of molecules of about 45,000 and 12,000 daltons both of which corresponded with molecules which were observed when NP40 extracts of virus-infected or uninfected cells were precipitated with anti-H-2 serum. Sequential immunoprecipitation analysis of infected cell extracts showed that S antigens were coprecipitated with either H-2K or H-2D antigens. These results suggest that the S antigens are somehow associated with H-2K or H-2D antigens separately.

The type of effector cells in immune spleen cells (ISC) responsible for cell-mediated cytolysis of mouse adenovirus-infected cells was characterized. When ISC were pretreated with anti-mouse thymocyte or anti-Thy1.2 serum and complement, their cytolytic activity assayed by the 51 Cr release test was completely abolished, whereas it was not significantly reduced either when ISC were pretreated with anti-mouse immunoglobulin serum and complement or after ISC were kept standing at 37°C for 2 h in a plastic dish to remove adherent cells. These data indicate that the effector cells in ISC are T cells. The release of nonspecific or specific soluble cytotoxic factor(s) from ISC upon contact with mouse adenovirus-infected cells was not demonstrated, but about 30 U of interferon activity per ml was detected. However, 10 times as many units of exogenous interferon or more showed no effect on 51 Cr release from target infected cells or on the inhibition of viral antigen synthesis in infected cells. Experiments with mouse cells of different major histocompatibility antigens indicated that the cytotoxicity of ISC is effective when sensitizer and target cells share all or part of the same H-2 gene complex but not effective when they are of a different H-2 gene complex.

Migration of peritoneal exudate cells (PEC), which were prepared from mice immunized against mouse adenovirus (M-Ad), was inhibited upon exposure to the antigenic extract of M-Ad-infected cells. This inhibition was shown to be blocked when infected cells or their extracts were pretreated with antiserum against M-Ad-induced cell surface (S) antigen(s) or with antisera against alloantigens of infected cells. Immune spleen cell-mediated cytolysis of M-Ad-infected cells was also blocked in the presence of anti-S, anti-alloantigen or anti-beta 2m serum. Immunofluorescent antibody staining of S antigen(s) was blocked when infected cells were pretreated with anti-alloantigen or anti-beta 2m serum, whereas it was not blocked when they were pretreated with anti-mouse immunoglobulin or anti-Thy-1.2 serum. Conversely, immunofluorescent antibody staining of alloantigens was blocked when infected cells were pretreated with anti-S serum. These findings indicate that S and alloantigens are associated with each other or at least located very close to each other.

A virus-specific cell surface antigen(s) was detected on mouse adenovirus-infected cells by the immunofluorescent antibody technique. It was demonstrated in infected cells regardless of the mouse strain or animal species used. It became detectable on the cell surface about 6 h postinfection and was demonstrated in 90% of infected cells at 24 to 36 h postinfection. On the other hand, intracellular capsid antigens became detectable by the immunofluorescent antibody technique at about 24 h postinfection. Since it was also detected on virus-infected cells treated with 5′-fluorodeoxyuridine to block viral deoxyribonucleic acid synthesis, it was synthesized at the early stage of infection and may not be a virus structural component. The relationship between this serologically detectable cell surface antigen(s) and cell-mediated immune response to virus infections is discussed.