Adenoviruses from human immunodeficiency virus-infected individuals, including two strains that represent new candidate serotypes Ad50 and Ad51 of species B1 and D, respectively.

Adenovirus (Ad) isolates from a large number of human immunodeficiency virus (HIV)-infected individuals were compared serologically and genetically with Ad isolates from immunocompetent patients. Between 1982 and 1994, stool and urine samples from 137 subjects with AIDS hospitalized in The Netherlands yielded 143 Ad strains. Forty additional Ad strains were obtained from 35 HIV-positive patients in Manchester, United Kingdom, in 1992 and 1993. Of these 183 HIV-associated Ad strains, 84% belonged to species D and 3% belonged to species C. These strains were compared with 2,301 Ad strains collected during general diagnostic examinations in The Netherlands from 1973 to 1992. Of the latter strains, 5% belonged to species D and 49% belonged to species C. Two of the Ads isolated from fecal specimens of AIDS patients represent new serotypes: candidate Ad serotype 50 (prototype strain, Wan) of subspecies B1 and candidate Ad serotype 51 (prototype strain, Bom) of species D. The DNA restriction enzyme patterns of strains Wan and Bom differed from the patterns of all established prototypes.

Rapid subgenus identification of human adenovirus isolates by a general PCR.

In most clinical situations involving adenovirus infection, subgenus (subgroup) identification of an adenovirus isolate is as informative as a finer identification by serotype. A PCR method which allows the identification of human adenovirus isolates as members of subgenera A, B:1, B:2, C, D, E, or F is described. It is based on a simple (nonnested) PCR using primers which bind to regions immediately flanking the VA RNA-encoding regions of human adenovirus genomes. The PCR allows amplification of DNA from all 49 human adenovirus prototype strains so far described. Since there are differences in the lengths of the VA RNA-encoding regions in adenoviruses of different subgenera, it is possible to differentiate some subgenera according to the size of the PCR product determined by electrophoresis. This forms the basis of an initial broad categorization of isolates as belonging to either (i) subgenus B:1, C, D, or E or (ii) subgenus A, B:2, or F. Subgenus identification is completed by a one-step restriction enzyme digestion and gel electrophoresis. The method was assessed by blind subgenus identification of 200 miscellaneous primate adenovirus isolates prepared by the reference laboratory at Bilthoven, The Netherlands. Identification at the subgenus level by PCR correlated 91.5% with the results of serotyping. A further 5.5% of isolates were correctly identified as belonging to one of two specified subgenera. Six of the 200 identifications (3%) were unsuccessful for various reasons, including weak PCR products, intermediate strains, and mistaken primate host. The method should serve as a rapid means of confirming adenovirus cytopathic effects in laboratories performing virus culture, with simultaneous subgenus identification of the isolate. It will also have relevance as an aid to conventional serotyping for epidemiological purposes, since for all adenoviruses except those belonging to subgenus D, neutralization tests need only involve a maximum of four type-specific antisera.

Detection, typing, and subtyping of enteric adenoviruses 40 and 41 from fecal samples and observation of changing incidences of infections with these types and subtypes.

Monoclonal antibody (MAb) preparations specific for the enteric adenoviruses of subgenus F (AdF) were generated and evaluated as typing reagents in virus neutralization tests and enzyme-linked immunosorbent assays (ELISAs). A panel of 11 genome types of adenovirus 40 (Ad40), 24 genome types of Ad41, and 47 adenovirus prototype strains was used to determine the specificities of the MAbs in the two assays. In this way two MAbs, MAb 40-1 (anti-Ad40) and MAb 41-1 (anti-Ad41) were selected. These two MAbs showed strict type specificity in both assays. A third MAb reacted in an ELISA with all 47 human adenovirus types. With two other MAbs, three antigenic subtypes of Ad41 could be distinguished by their reactivities in virus neutralization tests and ELISAs. On the basis of the five selected MAbs, a sensitive ELISA system was developed for the direct detection and simultaneous typing and subtyping of Ad40 and Ad41 present in stool specimens. The five MAbs were also used to study the epidemiology of infections with Ad40 and Ad41 in The Netherlands in the period 1981 through 1989. It was shown that there were no significant fluctuations in the annual incidence of the cluster of enteric adenoviruses as a whole. This cluster should therefore be considered to belong to the "endemic" rather than the "epidemic" adenoviruses. The relative incidence of Ad40 infections compared with that of Ad41 infections changed considerably during the period studied; the proportion of Ad41 infections rose from about 30% in 1981 to about 95% in 1986, after which it stabilized at 90 to 95%. The proportion of one of the subtypes of Ad41 (Ad41 subtype M3) increased from about 40 to 80% in the same period.

New developments in the molecular epidemiology of adenovirus 8 keratoconjunctivitis.

Four consecutive epidemics of keratoconjunctivitis caused by adenovirus 8 (Ad8) occurred over a 5-year period in Brest, France. A selection of 30 strains isolated during this period was studied by DNA restriction enzyme analysis using nine restriction enzymes. BglI and SacI were the most discriminative enzymes and allowed the recognition of four DNA variants, all different from the prototype strain Trim. Within each of the epidemics, the strains tested could not be distinguished in this analysis. Between strains from different epidemics differences in DNA structure could be detected however. Thus, the Ad8 epidemics of 1983/1984, 1984, 1987, and 1988 appear to have been due to DNA variants Ad8/D7, D8, D9, and D10, respectively. These results demonstrate that the DNA of Ad8 seems to display a considerable variability, comparable to that observed with Ad7 and Ad21. As has been described for Ad7, Ad21 and Ad41, successive DNA variants of Ad8 prevail during one or more years, and are then replaced by other, newly emerging variants sometimes associated with epidemics.

Characterization of fastidious adenovirus types 40 and 41 by DNA restriction enzyme analysis and by neutralizing monoclonal antibodies.

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.

Genome type analysis of adenovirus 37 isolates.

Seventy-six strains of adenovirus type 37 (AV37), isolated during a period of 10 years from 73 patients on three continents, were analysed with eight DNA restriction endonucleases. Strains identical with the prototype (56 strains) prevailed; four deviating genome types were found, which differed only slightly from the prototype, with one exception. Half of the strains were tested in detail by neutralisation and haemagglutination inhibition. Minor differences in neutralisation were not reflected by differences in DNA restriction patterns. In comparison to genome types of other subgenera, field strains of adenovirus 37 showed only a moderate genetic variability.

An epidemic of keratoconjunctivitis due to adenovirus type 37.

An epidemic of keratoconjunctivitis due to adenovirus type 37 in Liverpool in 1984 is reported. Initially serum neutralisation suggested that isolates were type 10 but further neutralisation studies supported by DNA restriction enzyme analysis showed that they were type 37. Clinical and epidemiological features of this cause of epidemic keratoconjunctivitis, recently recognised in the United Kingdom, are presented and the implications for the laboratory investigation discussed.

Molecular epidemiology of adenovirus type 21 in the Netherlands and the Federal Republic of Germany from 1960 to 1985.

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.

Adenovirus 38: a new human adenovirus species of subgenus D.

From the stool of a Dutch girl, a new virus (LJ) was isolated that by morphological, biological, and immunological criteria was a human adenovirus. Because of its hemagglutinating activity for rat and human red blood cells and its DNA restriction enzyme pattern, the virus was classified as a member of subgenus D. It was distinct in neutralization tests from all other human adenoviruses. The hemagglutinin was antigenically similar to that of adenoviruses 13 and 39, but the DNA differed from that of these two species. It is proposed to call virus LJ candidate Mastadenovirus h 38. Two other viruses belonging to the same new species were isolated in the USA.

Candidate adenoviruses 40 and 41: fastidious adenoviruses from human infant stool.

About 200 antigenically related adenoviruses were isolated from cases of infantile diarrhoea in the Netherlands and North-West Germany. The viruses were fastidious and failed to replicate serially in human diploid fibroblasts and in primary human embryonic kidney cells. A number of strains were established in HeLa, HEp-2, Graham (293), cynomolgus monkey kidney, and Chang conjunctival cells. The viruses were mammalian adenoviruses by the usual criteria. No relationship to the 39 known human adenovirus species was found, either by neutralization tests or by haemagglutination inhibition tests. Neutralization tests showed two distinct variants, represented by strains Tak and Dugan. The variants were identical in haemagglutination inhibition tests. DNA restriction enzyme analysis showed Tak and Dugan to have considerably different genomes, indicating that these variants should be classified as different species (Wadell et al, 1983). It is proposed that the variants should be called Mastadenovirus h 40 (with reference strains Dugan and Hovi X) and Mastadenovirus h 41 (with reference strain Tak). Neutralization and haemagglutination inhibition tests demonstrated that the viruses from Glasgow and Helsinki (Hovi X) described by Johansson et al [1980] and by Kidd and Madeley [1981] belong to these two adenovirus species.

Adenovirus 37: identification and characterization of a medically important new adenovirus type of subgroup D.

A new human adenovirus has been isolated from 62 eyes with (kerato)conjunctivitis and from nine genitourinary sites. The virus is closely related in haemagglutination inhibition tests to adenovirus type 19 (Ad 19) and Ad 10. Antiserum adsorption experiments demonstrated the presence of three haemagglutinin antigens in the virus: One unique, another common to Ad 19, and a third common to Ad 10 and 19. In neutralization tests, the virus is distantly related to Ad 13, 30, 19, and 10. Despite this relationship, it is proposed to call the virus adenovirus 37, in agreement with current species definitions. It belongs to subgroup D of human adenoviruses. Antisera to the new virus show virtually no neutralization of other human adenovirus types. Only bay use of this antiserum it is in practice possible to avoid wrong or indefinite typing, which has often occurred in the past.

Pharyngoconjunctival fever caused by adenovirus type 19.

A case of laboratory-acquired type 19 adenovirus infection is reported. The patient showed the classical triad of pharyngitis, acute follicular conjunctivitis and fever. Adenovirus type 19 was isolated from the affected eye, and the infection was confirmed by demonstration of seroconversion to this virus.