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Understanding the pathogenesis of viral myocarditis is linked to the availability of sensitive assays to detect viruses in clinical material. Recent advances in molecular techniques permit direct detection of viral-specific nucleic acid in tissue samples. This report describes a protocol for DNA extraction and amplification of adenovirus genome from formalin-fixed, paraffin-embedded human tissues that detects as little as 10 copies of viral genome in a background of 0.5 micrograms of human DNA. This sensitive assay permitted the examination of archived tissues to establish a retrospective diagnosis of adenoviral myocarditis in two pediatric patients.

Adenovirus isolates from 52 patients with ocular infection over a 3-year period were typed by restriction endonuclease analysis in a clinical laboratory. The results indicated that adenovirus type 8 was the most common cause of adenovirus eye infection during this period, being responsible for 42 (81%) of the 52 cases. Of 42 adenovirus type 8 isolates, 22 showed variant patterns by restriction endonuclease analysis and required multiple enzyme digests for identification. These isolates were readily identified by neutralisation tests.

Nosocomial transmission of adenovirus type 3 associated with fatalities in infants has not been frequently reported. This report describes the nosocomial spread of adenovirus types 2 and 3 among infants with bronchopulmonary dysplasia in a chronic (transitional) care facility. The index case developed pneumonia with a clinical deterioration in respiratory status 8 days after admission. Within the next 10 to 30 days 9 other infants and 2 health care personnel became ill with respiratory symptoms. Three of these 10 infants had progressive respiratory failure and 2 of them died. All of these infants had underlying chronic lung disease of bronchopulmonary dysplasia. The overall attack rate was 30% (10 of 33). Further spread of adenovirus was prevented by using barrier precautions and masks while performing tracheostomy care. Adenovirus isolates were serotyped as Ad3 in 4 patients and 1 staff member, as Ad2 in 3 patients, and as a combination of Ad2 and Ad3 in 1 patient. Two fatalities were associated with Ad3 infection. Three isolates from 2 patients and 1 staff member were not available for typing. Restriction endonuclease analysis was performed on all of these isolates of Ad3 and Ad2. There was no genetic heterogeneity in the isolates, suggesting a common source.

The polymerase chain reaction (PCR) was used to detect adenovirus DNA in formalin-fixed, paraffin-embedded tissue obtained post mortem. Adenovirus DNA was successfully amplified from specimens of lung and liver from two patients with disseminated adenovirus infection confirmed by virus isolation, electron microscopy and/or immunohistochemistry. Negative results were obtained for specimens of lung from two patients with cytomegalovirus pneumonia. The specificity of the adenovirus PCR was confirmed by means of a digoxigenin-labelled probe generated in a separate PCR. Detection of viral nucleic acid by PCR in tissues obtained post mortem has considerable diagnostic potential.

Formation of progeny viruses in the nuclei of HeLa cells infected with adenovirus type 5 was studied at the ultrastructural level by in situ hybridization techniques allowing specific detection of either viral double-stranded DNA (dsDNA) or single-stranded DNA (ssDNA). Prior to the initiation of replication of viral genomes, infective DNA molecules which entered the nucleus of the target cell were randomly distributed among host chromatin fibers including nucleolus-associated chromatin. They were double-stranded, that is, without single-strand breaks. Such association of viral DNA with host condensed chromatin also occurred in mitosis. The initiation of viral genome replication occurred simultaneously with the appearance in the nucleoplasm of small fibrillar regions containing intermingled viral dsDNA and ssDNA. Later, at the intermediate stage of nuclear transformation, viral dsDNA and ssDNA molecules were almost entirely separated into two contiguous substructures. At this stage, viruses were observed occasionally in the vicinity of viral ssDNA accumulation sites. Still later, an additional substructure developed in the centre of the nucleus which consisted of large quantities of viral dsDNA, traces of viral ssDNA and abundant viruses. Portions of viral ssDNA were attached to some viruses even at late stage of nuclear transformation, an association which strongly suggests the occurrence of encapsidation of at least some of the viral genomes while they are still engaged in replication.

Adenovirus (Ad) type 40 and 41 DNAs were directly extracted from stool specimens of children with gastroenteritis. Two new strains of Ad41, Sanekata and Ehime strain, were cloned and their restriction maps were constructed. The left terminal end of the cloned Ad41 genome, EcoRI-E fragment of the Sanekata strain and EcoRI-F fragment of the Ehime strain, had transforming ability in rat 3Y1 cells. Only one of the 35 isolates of Ad40 tested showed a different restriction profile, while three different restriction profiles were found in DNAs from Ad41 isolates.

Adenovirus type 31 (Ad31) was isolated from 15 immunocompromised patients in 12 of whom seroconversion was also recorded. Ad31 infection has a substantial clinical relevance since 8 of 10 with lower respiratory tract infection and 4 of 4 with hepatitis died. Therefore, Ad31 isolates from immunocompetent and immunodeficient hosts were compared by restriction endonuclease analysis. Nine genome types were identified among the 79 Ad31 isolates. Pairwise comparison of comigrating restriction fragments indicated that the genome types could be divided into three genomic clusters. Several Ad31 genome types were isolated from immunocompromised patients, but no highly virulent genome type could be found. A genome type was identified in a child with severe combined immunodeficiency who originally was infected with another genome type. This observation is suggested to have evolutionary implications.

A cluster of adenovirus type 5-caused acute respiratory diseases was observed in Prague in the autumn-winter season of 1978/79. An analysis of viral DNA by sequence-specific restriction enzymes in two out of 39 isolates revealed that these isolates, identical to each other, were clearly distinct from adenovirus type 5 prototype strain. The designation "adenovirus genome type 5a" is suggested for these isolates.

Enteric adenoviruses (EAds) (candidate adenoviruses 40 and 41, subgroups F and G) have been implicated in the etiology of gastroenteritis in infants, but their clinical significance has been unclear because a rapid test to distinguish these agents from other adenovirus (Ad) types has not been available. We developed a dot-blot hybridization assay for EAd DNA using a cloned DNA fragment that has little homology to non-EAd DNAs. The dot-blot system detected less than 20 pg of EAd DNA, while showing minimal cross hybridization to representative strains from all other Ad groups. There was no detectable hybridization to extracts of samples known to contain other enteric viruses. It was further shown that low levels of EAds in specimens could be amplified by culturing for 1 day in 293 cells. Stool samples and tissue culture lysates prescreened by electron microscopy, cell culture or ELISA were tested in a blind fashion. Using endonuclease analysis as the standard for typing the isolates, we found the dot-blot system to have a 91% sensitivity and 71% specificity for detecting EAds and distinguishing them from other Ads. False-positive and equivocal dot-blot results appeared to be caused by other Ads.

Restriction endonuclease analysis was carried out on adenovirus types 3, 4, and 7 (Ad3, Ad4, and Ad7, respectively) isolated during a 16-month epidemic period. Most of the isolates were associated with respiratory or ocular infection or both. Forty-four strains of the subtype Ad7b were identified with SmaI; these strains could be further subdivided into three distinct genome groups, designated Ad7b1, Ad7b2, and Ad7b3, by digestion with PvuII, BglI, and SstII. These Ad7b variants occurred at approximately similar frequencies throughout the epidemic and were associated with similar clinical illnesses. Nine distinct intratypic genome groups were identified among 29 Ad3 strains with HindIII, PstI, and SmaI; adenoviruses similar to subtype Ad3a, formerly reported to be uncommon in Europe, were isolated most frequently (41% of strains), and the previously predominant prototype (Ad3p) was not found. Considerable similarities among the genomes of some strains of Ad3 and Ad7 could be demonstrated, and clinical and epidemiological presentations were very similar. The four strains of Ad4 isolated fell into two distinct patterns, i.e., the previously recognized prototype (Ad4p) and a new subtype designated Ad4b. Both of these subtypes were associated with follicular conjunctivitis. Thus we were unable to produce evidence that particular genome types of Ad3, Ad4, and Ad7 are associated with particular clinical presentations. However, restriction endonuclease analysis should prove useful for epidemiological studies on these viruses.

The function of the adenovirus-coded terminal protein and its precursor in viral DNA replication was studied by raising an antiserum against the adenovirus type 5 (Ad5) terminal protein isolated from virions. This antiserum reacted with both the terminal protein and its precursor as measured by a radioimmunoassay. In an in vitro DNA replication system employing nuclear extracts the addition of antiserum inhibits replication when a DNA-terminal protein complex from adenovirions is used as template. The replication of a 3.8% terminal fragment of the Ad2 genome with a protein-free origin (derived from the plasmid XD-7) is also inhibited by the antiserum. This observation confirms a role of the terminal protein precursor in DNA replication. The antiserum completely inhibited the formation of a covalent complex between the precursor terminal protein and dCMP, which is essential for initiation. A function of the terminal protein in the elongation reaction was shown by the inhibitory effect of antiserum on DNA chain elongation in isolated nuclei from Ad5-infected cells. Also in the in vitro DNA replication system employing nuclear extracts the elongation reaction is strongly reduced by addition of the antiserum. These results indicate that the terminal protein and/or its precursor are not only involved in initiation of DNA replication but also in DNA chain elongation.

We studied the restriction endonuclease cleavage patterns of DNAs of adenovirus type 8 (Ad 8) isolated from epidemic keratoconjunctivitis cases. DNAs of 25 Ad 8 isolates collected during the period from 1975 to 1981 in Sapporo were subjected to enzymatic cleavage with PstI, BamHl , HindIII and SalI. On the basis of the cleavage patterns, the isolates were divided into two subtypes, A and B, both different from Trim strain used as the Ad 8 prototype strain. Subtype A was prevalent in the period from 1975 to 1978, while subtype B in the period from 1976 to 1981.

During an epidemiological survey made in São Paulo (Brazil), fecal specimens were periodically collected from 100 randomly chosen babies from birth to the age of 18 months. The stools, routinely collected each month and also collected each time a child presented any sign of disease, were screened for the presence of adenoviruses. Sixteen adenovirus strains, isolated from the stools of healthy and ill children, were characterized by restriction enzyme analysis. Five isolates were from subgenus A, five were from subgenus B, four were from subgenus C, and two were from subgenus D. All but two showed some restriction patterns different from those of the 42 human adenovirus prototypes and all the genome types described up to now. No fastidious adenovirus (subgenus F, serotypes 40 and 41) was encountered in the stools examined. We report here the restriction enzyme analysis of isolates of subgenera B and C. The following new designation genome types are proposed: Ad3e1 (subgenus B) and Ad1d, Ad5a1, and Ad5a2 (subgenus C).

Adenovirus type 8 strains were collected over a 19-year period from eye specimens from patients with keratoconjunctivitis. These strains were divided by restriction enzyme analysis with the endonucleases SalI, HindIII, SacI, KpnI, and SmaI into three genotypic subgroups. The prototype strain (Trim) was found throughout the United States from 1966 through 1985 and also in Taiwan and Greece in the early 1980s. Genotype 8C was identified in an arc from Maryland to Missouri to Alabama from 1971 through 1974. Genotype 8D was found only in an epidemic of eye disease among Vietnamese refugees being resettled in northwest Florida in 1975. Genotypes 8C and D were distinct from genotypes 8A and B described from Japan in 1975 through 1981. Hemagglutination tests with a battery of avian and mammalian erythrocytes did not distinguish between the genotypic subgroups. Similarly, the genotypes could not be differentiated by hemagglutination inhibition or serum neutralization tests with reference prototype antisera. The long-term prevalence of the Trim strain suggests that adenovirus type 8 has greater genetic stability than the other adenovirus types studied to date.

The DNA of 19 wild strains of adenovirus types 10 (AV10), 13, 19, 24, and 27 was analysed by five restriction endonucleases (BamHI, BglII, BstEII, HindIII, and SmaI) for their genetic variability and compared with their respective prototypes. Six "AV19a" strains were identical among each other, even with five additional enzymes, but widely different from the prototype. Strains of AV10, 24, and 27 were identical or only slightly different from the respective prototypes. In contrast, three AV13 strains differed in all enzyme patterns from those of the prototype and from one another.

Several of the 41 types of human adenovirus have been divided into genome types based on aberrant restriction endonuclease digestion patterns of viral DNA. In the process of screening a large number of clinical adenovirus isolates by restriction endonuclease digestion of viral DNA, we have identified nine isolates from eight patients which were identified by neutralization as adenovirus type 1 or 2 but had aberrant cleavage patterns. Cleavage sites for the enzymes SmaI, EcoRI, HindIII, KpnI, and HpaI were mapped. The variants could be placed in six distinct groups based on cleavage patterns. The designation genome types 1a, 1b, 1c, 2b, 2c, and 2d are proposed for these isolates.

We have used a probe against the adenovirus genome to study cultured epithelial cells specifically infected with various types of adenovirus and Graham 293 cells, which contain few copy numbers of a fraction of the adenovirus genome. We have also examined lung tissue obtained from three cases of acute adenovirus pneumonia, two cases of adenovirus pneumonia that had passed through the acute phase, and nine cases of follicular bronchiectasis. Our purpose was to determine whether the probe was effective in detecting a wide variety of adenovirus types, to determine whether it could detect adenovirus in lung tissue that had been fixed and stored in paraffin blocks for several years, and to test the hypothesis that adenovirus was an important cause of follicular bronchiectasis. The results show that the probe was able to detect adenovirus from Genera B1, B2, C, D, and E with a sensitivity of 5 to 10 copies/cell. The probe also detected adenovirus in 14 of 14 slides from three cases of acute disease, but failed to obtain a positive result in the cases examined after an acute infection or in any of the cases of follicular bronchiectasis. We conclude that the in situ hybridization technique is useful in the investigation of active adenovirus infection of the lung. The failure to show that the virus persisted in the chronic respiratory disease that follows adenovirus infection, or that it was present in cases of follicular bronchiectasis could be due to either a true absence, or to its presence in a latent form that is below the level of sensitivity of this technique.

Flow cytophotometry and time-lapse cinematography were used to study viral DNA synthesis and alteration of the cellular DNA content of human cells infected by adenovirus type 2 wild-type and two DNA-negative temperature-sensitive mutants. Cell populations with DNA contents greater than 4n (where n represents the normal haploid DNA content of cells) were found after infection and their origin is discussed with regard to cell cycle changes, viral DNA replication and cell fusion or endomitosis.

DNA cleavage analyses with EcoRI, HindIII and BamHI were carried out to investigate genome types of recent Ad 4 isolates obtained from acute respiratory disease (8 strains), and ocular disease (11 strains) in Japan. DNA cleavage patterns of all 19 isolates studied were identical regardless of whether they were recovered from respiratory tract or conjunctiva, but were distinct from that of the prototype strain.

The DNA of 48 strains of adenovirus type 40 (Ad40) and of 128 strains of adenovirus type 41 (Ad41), isolated between 1971 and 1986 from various countries, was characterized by restriction enzyme analysis using nine and ten restriction endonucleases respectively. Five new DNA variants of Ad40 and 18 new DNA variants of Ad41 were detected. Most of the restriction sites which differed among the various DNA variants appeared to be distributed at random over the entire length of the viral genomes of the two serotypes. The number of restriction sites by which two DNA variants differed from each other was used as a measure of their relatedness. Several clusters of closely related DNA variants were observed for each of the two serotypes. The 35 DNA variants of Ad40 and Ad41 were used to test monoclonal antibody preparations for their range of reactivity in a neutralization assay. One monoclonal antibody (5-8), raised against Ad40 strain Dugan, showed type-specific neutralization of all 11 Ad40 DNA variants tested. Six monoclonal antibodies, raised against Ad41 strain Tak, neutralized different proportions of the variants of Ad41. Two of these preparations (1-21 and 3-19) neutralized all 24 Ad41 DNA variants, while a third (1-23) reacted with only 12 Ad41 variants. Three other monoclonal antibody preparations (3-10, 3-18, 7-14) reacted specifically with only 6 of these 12 variants. The patterns of reactivity with the monoclonal antibody preparations correlated with the presence or absence of a HindIII restriction site at 56 map units and of an EcoRI restriction site at 52 map units on the Ad41 DNA. This region of the adenovirus DNA codes for the hexon protein, which is known to contain the type-specific neutralizing antigenic determinants.

One hundred thirty-eight strains of adenovirus type 3 isolated from patients in the United States, West Germany, and other regions between 1961 and 1984 were analyzed with six restriction endonucleases; 18 genome types were found. BglII was the most discriminative enzyme. Mapping of altered restriction sites was also performed for all six enzymes. The genome types D1 (like the prototype) and D3 prevailed among 45 and 47 strains, respectively. All genome types could be divided into two groups related to D1 or D3. Several clusters of infections by strains with the same genome type were observed. Only hints of differences were found in the pathogenicities of individual genome types. D1 strains were present in the United States and in Europe; group D3 prevailed almost exclusively in the United States.

After a period of high prevalence in the early 1960s, adenovirus serotype 21 (Ad21) was identified in The Netherlands only very sporadically for more than 20 years. From December 1984 to July 1985, Ad21 was isolated relatively often from hospitalized children living in different parts of The Netherlands. The patients in question suffered from respiratory, gastrointestinal, meningeal, or ocular disorders. An increase in the incidence of Ad21 infections was also observed in the Federal Republic of Germany during this period. The DNAs of 93 isolates of Ad21 were subjected to restriction enzyme analysis with eight endonucleases. All 50 strains isolated in The Netherlands between 1960 and 1963 proved to be DNA variant Ad21/D2/20655/Netherlands/60. This variant has already been described (T. Adrian, R. Wigand, and J. C. Hierholzer, Arch. Virol. 84:79-89, 1985) as typical for the Ad21 strains circulating since 1960. Analysis of the DNAs of the 28 Ad21 strains isolated in The Netherlands or in the Federal Republic of Germany in 1984 and 1985 showed them to belong to two new, closely related DNA variants designated Ad21/D7/1857/Netherlands/84 and Ad21/D8/5398/Netherlands/85. The BglI and KpnI restriction profiles were characteristic for these recent DNA variants.