Selection of nonfastidious adenovirus species in 293 cells inoculated with stool specimens containing adenovirus 40.

Of 35 stool specimens isolated and examined in 293 cells, 15 isolates contained adenovirus species 40 (Ad40), and 4 of these 15 isolates also contained a nonfastidious adenovirus species (Ad1 in two cases, Ad18 or Ad31) which was selected over Ad40 during serial passage in the 293 cells. The selection of Ad1 over Ad40 was examined in detail. Restriction analysis of intracellular DNA and the relative infectivity titers of Ad40 and Ad1 at each passage level after the inoculation of 293 cells with a particular stool specimen demonstrated that although the amount of Ad40 DNA synthesized far exceeded that of Ad1, the relative infectivity titer of Ad40 was low. The growth characteristics of Ad40 were then compared with those of Ad1, Ad18, and Ad41 in singly infected 293 cell cultures. One-step growth curves showed the same growth rate in each case, with a latent period of 12 h and a maximum titer at 24 to 36 h postinfection. Yields of infectious Ad40 virus were consistently 100- to 1,000-fold lower than those of Ad1. This difference was reflected by a reduced yield of total AD40 virions (p1.34) as determined by 35S labeling experiments. However, the 3- to 10-fold reduction in total yield of Ad40 virions did not account for the 100- to 1,000-fold reduction in the yield of infectious virus.     

Isolation and propagation of enteric adenoviruses in HEp-2 cells.

Eighty-two stool samples from children with gastroenteritis in Canada, England, and Thailand which had been shown to contain adenovirus antigen (by a group-specific enzyme-linked immunosorbent assay) or adenovirus particles (by electron microscopy) or both, were tested for primary isolation of enteric adenoviruses in HEp-2 and Graham 293 cells. Graham 293 cells are known to support the replication of enteric adenovirus types (Ad40 and Ad41) on primary isolation, whereas HEp-2 cells reportedly do not. Of the 82 adenovirus isolates, 73 could be typed as Ad40 or Ad41 by type-specific monoclonal antibody enzyme-linked immunosorbent assay and by analysis of SmaI endonuclease digests. Of these 73, 30 (41%) could be isolated in HEp-2 cells, which included 43% (9/21) of those typed as Ad40 and 40% (21/52) of those typed as Ad41. On the basis of these results, the growth characteristics of adenoviruses in HEp-2 cell cultures, commonly used to distinguish enteric from nonenteric adenovirus types, are not valid for either diagnosis or epidemiological studies. For the samples studied here, use of these nondefinitive criteria would result in underestimation of the incidence of enteric adenoviruses in viral gastroenteritis.     

Prevalent enteric adenovirus variant not detected by commercial monoclonal antibody enzyme immunoassay.

A commercial monoclonal antibody enzyme immunoassay for the detection of enteric adenovirus type 40 and 41 (Ad40 and Ad41) in stool specimens was evaluated. Twenty-one stool specimens from children with gastroenteritis, with adenovirus particles visible by electron microscopy, and reference strains Ad40 Dugan and Ad41 Tak were tested by Ad40- and Ad41-specific and adenovirus group-reactive immunoassays. All stool specimens tested positive in the group-reactive immunoassay. However, only six specimens, containing isolates of Ad40 strain Hovi-X, an Ad40 genomic variant, and Ad41 strain Tak, reacted with the specific immunoassay, besides the reference strains. Fifteen stool specimens determined by restriction analysis to contain a genomic variant of Ad41 were negative by specific immunoassay. The positions of restriction site differences from the prototype strain Ad41 Tak were analyzed, and four mutations were mapped within the hexon gene; two others may occur in the fiber gene. The Ad41 genomic variant not detected by the enteric test is presently the most frequent cause of local adenoviral gastroenteritis. Highly specific monoclonal antibodies can fail to detect genomic variants of enteric adenoviruses, probably because of alteration of external neutralizable epitopes under immunological pressure to vary.     

Specific detection and typing of adenovirus types 40 and 41 in stool specimens by dot-blot hybridization.

A dot-blot hybridization test was developed which allowed the direct detection of fastidious enteric adenovirus DNA in stool specimens from children with diarrhea and simultaneous typing of the viruses as adenovirus type 40 (Ad40) or Ad41. Cloned PstI fragments of Ad40 and Ad41 were used as 32P-labeled probes in the test, which allowed detection of picogram quantities of viral DNA in 20 to 40 microliter of stool suspension. Results were obtained within 48 h. The type specificity of the test was evaluated with 76 specimens known to contain either Ad40 or Ad41 by restriction enzyme analysis. Sixty-one specimens had sufficient DNA to be detected without any removal of protein. Thirty-one adenoviruses were typed as Ad40, and 30 were typed as Ad41, giving 100% correlation with the results of restriction enzyme analysis. The other 15 specimens were detected and typed as Ad40 or Ad41 only after removal of protein by a phenol extraction method. The dot-blot hybridization method is particularly useful for identifying those Ad40 and Ad41 strains which defy all attempts at culture and will be a useful tool in the epidemiology of fastidious enteric adenovirus infections.     

Propagation of adenovirus types 40 and 41 in the PLC/PRF/5 primary liver carcinoma cell line.

The sensitivity of cell cultures to adenovirus types 40 and 41 (Ad40/41) was compared by means of cell culture infectious dose (ID50) assays using monolayer cultures in microtitre plates. The PLC/PRF/5 cell line derived from a primary human hepatocellular carcinoma was 100 times more sensitive to a laboratory strain of Ad41, and 10 times more sensitive to a laboratory strain of Ad40 and two Ad41 stool isolates, than Graham 293 and Chang conjunctival cells commonly used for the propagation of these viruses. In microtitre plate titration assays PLC/PRF/5 cells retained an optimal condition for longer and displayed cytopathogenic effects earlier and more clearly than the other cell lines. In contrast to previously used cells, PLC/PRF/5 cells also proved successful for the quantitation of Ad41, but not Ad40, by conventional plaque assays. The reason for the exceptional susceptibility of PLC/PRF/5 cells has not been elucidated, but the findings open attractive new doors for research on Ad40/41.     

Laboratory identification of adenoviruses associated with gastroenteritis in Canada from 1983 to 1986.

A retrospective analysis of adenovirus serotypes associated with gastroenteritis involved the examination of 143 stool specimens collected between 1983 and 1986 from symptomatic patients whose stools were positive for adenovirus by electron microscopy. The virus isolates obtained from 140 of the specimens were typed according to the SmaI cleavage pattern of the viral DNA and by neutralization with specific antisera. The predominant types were adenovirus type 31 (Ad31) (18%), Ad40 (16.9%), and Ad41 (38%), which together accounted for more than 70% of the isolates. The remaining virus isolates were typed as Ad1, 2, 3, 5, 7, and 12. DNA restriction analysis proved to be better than serum neutralization for identification of the enteric adenovirus serotypes in stool specimens. HindIII cleavage identified four Ad41 variants, none of which had a HindIII restriction pattern identical to that of the prototype strain Tak. Over the time period of the study, the incidence of Ad40 showed an overall decrease accompanied by an increased incidence of Ad41, while the incidence of Ad31 was relatively stable.     

Time-resolved fluoroimmunoassay for enteric adenoviruses using the europium chelator 4,7-bis(chlorosulfophenyl)-1,10-phenanthroline-2,9-dicarboxylic acid.

A time-resolved fluoroimmunoassay (TR FIA) was developed for the direct detection of adenovirus types 40 (Ad40) and 41 (Ad41) in stool specimens by using a monoclonal antibody (5D8/2C2) which recognizes both Ad40 and Ad41 but does not cross-react with other adenovirus serotypes. In this assay, the detector antibody is biotinylated directly rather than labeled with europium, and the fluorescent signal is generated on a solid phase in the presence of excess europium (Eu3+). The strength of the signal is dependent on the amount of a Eu3+ chelator [4,7-bis(chlorosulfophenyl)-1,10-phenanthroline-2,9-dicarboxylic acid (BCPDA)]-streptavidin complex bound by the biotinylated detector antibody (5D8/2C2). In a pilot study with 41 specimens, this TR FIA demonstrated a maximum sensitivity and specificity of 88% compared with SmaI restriction analysis of adenovirus isolates from the same specimens. TR FIA using the europium chelator BCPDA represents a feasible approach for the direct identification of specific adenovirus serotypes in stool specimens.     

Enzyme-linked immunosorbent assay for detection of enteric adenovirus 41

An enzyme-linked immunosorbent assay (ELISA) for direct detection of enteric adenovirus 41 (Ad41) in stool specimens was developed and compared with an Ad40-specific ELISA described previously [Johansson et al, 1980]. Rabbit antiserum to Ad41 was obtained by immunization with purified virions. To eliminate genus-specific reactivity the serum was passed through an immunosorbent column containing soluble adenovirus components of members of subgenera A to E. The anti-Ad41 serum still displayed high reactivity against Ad40 and had to be immunoabsorbed with soluble virus components of Ad40 to be rendered type-specific. The absorbed antiserum was used in an indirect ELISA and proved to be specific for Ad41. No heterotypic reactivity against Ad40 or Ad1 through Ad35 was found. The Ad41-specific ELISA proved to be of equal sensitivity to electron microscopy. The type-specific ELISAs for Ad40 and Ad41 were evaluated by testing 76 stool specimens containing enteric adenoviruses originating from England and Scandinavia. All specimens could be typed--41 (54%) as Ad40 and 35 (46%) as Ad41. These results were confirmed by DNA restriction site analysis. The type-specific ELISA proved to be a specific, sensitive, and a rapid technique for detection of Ad41 and allowed clear-cut discrimination from Ad40 in clinical specimens.     

Enteric adenovirus infection among infants with diarrhea in rural Bangladesh.

A total of 4,409 stool specimens from infants less than 5 years of age seeking treatment for diarrhea in Matlab, Bangladesh, were tested for the presence of adenoviruses by using an enzyme immunoassay (EIA). EIA-positive stool samples were serotyped with monoclonal antibodies specific for adenovirus type 40 (Ad40) and Ad41 and group antigen, inoculated into Graham G293 cells, and retested by EIA. Of adenovirus-positive cultures, 125 (2.8%) specimens were confirmed as enteric adenoviruses (EAds), of which 51 (40.8%) were typed as Ad40 and 74 (59.2%) were typed as Ad41, and 12 of 4,409 (0.3%) were identified as nonenteric adenoviruses. A slight peak of incidence of EAd infection was observed in the cool, dry months, and an outbreak of Ad40 infections occurred in March 1988, when the detection rate of EAd reached 12.3%. Information on age, gender, and symptoms was available for 80 infants infected with adenovirus only. Age distribution was similar for types 40 and 41 and nonenteric adenovirus; the median ages were 11, 12, and 12 months, respectively. The ratio of males to females for the 80 infants varied according to serotype; Ad40 had the highest male/female ratio, 2.17. The symptoms experienced by the 80 children were similar for each adenovirus type. The most common clinical features of EAd infection were watery diarrhea (87.5%), more than eight loose bowel movements per day in the 24-h period prior to presentation (68.8%), with vomiting (80.0%), abdominal pain (76.3%), and low-grade fever (95.0%); these symptoms are significantly similar to symptoms of infants infected with group A rotavirus. EAd infection generally gave rise to mild to moderate dehydration, which is significantly similar to dehydration produced by infection with rotavirus.     

Differential growth of human enteric adenovirus 41 (TAK) in continuous cell lines

Differing reports exist about the replication of human enteric adenoviruses (EnAds) in various cell lines. There was a suggestion that EnAds are defective, do not grow on primary human diploid cells, and behave like Ad host-range mutants, i.e., they require early gene products from other Ad types for efficient growth. Thus, initially the Graham-293 cell line, which contains the E1 region (E1A, E1B) of Ad5, was thought to be an ideal host for EnAds, because it provided the needed functions. Our findings, however, question this contention and show that Ad41 strain TAK, cultured on 293 cells, rapidly loses its infectivity (greater than 90% on the first and 100% by the second passage). In contrast to the results with 293 cells, we found that Ad41 strain TAK can be serially grown to high titers on several continuous cell lines: namely, HeLa, HI407, or HEp-2 cells. In order to investigate the basis for the rapid loss of the Ad41 infectivity upon passaging in 293 cells, Ad41 virions were purified from 293, as well as from HEp-2 cells, and their composition was analyzed. When structural proteins of the complete virions (rho = 1.34 g/cm3) were compared by Western blot analysis using polyclonal antibodies to HEp-2-grown virus, only traces of protein V (Mr 46,000 Da) could be detected in particles from 293 cells. In contrast, Ad41 particles obtained from HEp-2 cells exhibited a strong band at the position of protein V. Further, if polyclonal antibodies to 293-grown Ad41 were used in the Western blot, no protein V band was detected in HEp-2-grown virus. Finally, we note that new protein bands (Mr 25,000-35,000 Da) could be observed upon Western blot analysis of 293-derived complete and incomplete Ad41 particles. All of these observations taken together suggest that the low infectivity of Ad41 particles, prepared from 293 cells, could be due to a defect in assembly.     

Monoclonal antibody enzyme-linked immunosorbent assay for specific identification and typing of subgroup F adenoviruses.

Monoclonal antibody specific for subgroup F enteric adenoviruses (EAds) was prepared by fusing P3-NS1/Ag4-1 mouse myeloma cells with lymphocytes from BALB/c mice immunized with G1105, an adenovirus type 41 (Ad41) strain. Monoclone 3F11/2H9, which specifically recognized Ad41, was successfully used as detector antibody in an enzyme-linked immunosorbent assay (ELISA). Additionally, previously prepared monoclones 5D8/2C2 and 2H6/1E11, recognizing Ad40 plus Ad41 and Ad40 alone, respectively, were used to study stool and/or tissue culture specimens from 106 patients with adenovirus-positive gastroenteritis. By ELISA, 91 had EAds (22 were Ad40 and 69 were Ad41) and 15 had non-EAds. ELISA results were in concordance with restriction endonuclease results for 38 of 39 specimens, with dot blot data for 19 of 20 specimens, and with neutralization test results for 74 of 78 specimens. ELISA was at least 10-fold more sensitive than direct electron microscopy was for the detection of EAds in stool specimens.     

Incidence of enteric adenovirus gastroenteritis in Iranian children.

BACKGROUND: Enteric adenoviruses, i.e. adenovirus 40 (Ad40) and adenovirus 41 (Ad41), have been shown to be a substantial cause of pediatric gastroenteritis in various parts of the world, but no data are available for Iran. OBJECTIVE: The present study was performed to determine the incidence of enteric adenoviruses in children presenting to the Children's Medical Center with gastroenteritis in Iran. STUDY DESIGN: Stool specimens from 872 children less than 7 years of age attending the Children's Medical Center in Tehran, Iran, with gastroenteritis were tested for the presence of Ad40, Ad41, and adenovirus-genus by a monoclonal antibody-based enzyme immunoassay. RESULTS AND CONCLUSION: 6.7% of stool specimens contained enteric adenoviruses (3.3% Ad40 and 3.4% Ad41) and 2.0% nonenteric adenoviruses. Mean ages of Ad40, Ad41 and NEAd-positive children were 21, 19 and 29 months, respectively. Among the adenovirus-positive patients, 53.9% were male and 46.1% female. Watery diarrhea was present in 86.4% of children infected by adenoviruses. In conclusion, for the first time, we demonstrated the presence of enteric and nonenteric adenoviruses in a considerable proportion of stool samples from Iranian children with gastroenteritis.     

Enteric adenovirus 41 (Tak) requires low serum for growth in human primary cells

It had been postulated that due to lack of growth of enteric adenovirus 41 (Ad41) on human primary cells and its growth on Graham-293 cells there was a defect in the Ad41 E1A region. However, we found as a result of careful evaluation of Ad41 growth on several primary cell lines (HEK, WI-38, or Detroit 551) that efficient viral multiplication is possible if the serum concentration in the medium used postinfection (p.i.) is kept between 0.2 and 1%. In contrast, only slight growth of Ad41 occurs in infected cells maintained in 5% serum and virtually no viral replication is found in infected cells cultivated in medium with 10% serum. The serum inhibitory effect appears limited to primary cells because no difference in Ad41 replication, as assayed by accumulation of Ad41 DNA, was found in infected continuous cell lines (HEp-2, 293) cultivated p.i. in either 1 or 10% FBS. Also, this effect appears specific for enteric adenoviruses, such as Ad41, since conventional adenoviruses, such as Ad5, grow well in both 1 and 10% FBS. The above results show that Ad41 can grow in a variety of primary cell lines, under specific culture conditions. In addition, we found that Ad41-infected primary cells grown in medium containing 0.2% serum had an increase in synthesis of the 70-kDa heat shock protein (HSP70) at about 6 hr p.i. and also Ad41 was able to complement the Ad5 E1A deletion mutant dl312. These results show that the E1A function of Ad41 is not impaired in infected cells.     

Silver staining of DNA restriction fragments for the rapid identification of adenovirus isolates: application during nosocomial outbreaks

The ultrasensitive photochemical silver stain for nucleic acids, described by Beidler et al. (1982), has been applied to the detection of adenovirus restriction fragments as a relatively rapid technique for the identification of virus isolates. In this study, restriction enzyme cleavage analysis was used to characterize adenovirus isolates from what appeared to be two nosocomial outbreaks. The first outbreak was thus shown to include two clusters of patients, and involved two serotypes Ad7c and Ad40. The second outbreak was unrelated and involved Ad35. Although restriction analysis does not replace serum neutralization as a routine method for typing adenoviruses, it is a much more rapid means of discriminating between different patient isolates, providing a current rather than retrospective analysis of a nosocomial outbreak. During the first outbreak, restriction analysis identified two distinct adenovirus serotypes from one patient--Ad7c from a nasopharyngeal aspirate and Ad41 from a stool specimen. Restriction analysis is also valuable for the sub-typing of virus isolates. In this study, the Ad40 and Ad41 isolates were shown to be variants of the respective prototype strains.     

Physical organization of the enteric adenovirus type 41 early region 1A

Enteric adenovirus types 40 and 41 (Ad40 and Ad41), representing subgenus F, differ from all other human adenoviruses by being so fastidious that productive replication does not occur in conventional established cell lines. They are dependent of the Ad5 early regions E1A and E1B since they can not grow in HEK cells, only in 293 HEK cells transformed by Ad5 E1. The overall genetic organization of Ad41 E1A is similar to the E1A region of other characterized human adenoviruses but it is slightly shorter, comprising 1350 bp. The inverted terminal repeat (ITR) at the 5' end of both Ad40 and AD41 consists of 163 nucleotides, being similar to the ITR of Ad12 (subgenus A) and longer than the ITRs of adenoviruses of subgenera B, C, and E. The early mRNA products (12 and 13 S) can be translated into a 222-amino acid (aa) and a 251-aa tentative protein, respectively. In a comparison of the Ad41 251-aa protein with corresponding peptides of Ad12, Ad7, Ad5, and Ad4, three conserved amino acid sequences CS1-CS3 can be found. In the second conserved domain CS2, which is particularly acidic, the homology is very high within all five serotypes compared. Only one among eight conserved amino acids differs in the Ad41 251-aa protein. Within CS1 and CS3 which exhibit a hydrophilic and a hydrophobic character, respectively, the amino acid composition of the Ad41 protein is less conserved than the corresponding regions in all other analyzed adenovirus types. Ten of 16 conserved amino acids in CS1 are shared by Ad41 and 18 of 23 conserved amino acids in CS3 are shared by Ad41.     

Evaluation of cell line 293 for virus isolation in routine viral diagnosis.

Cell line 293, a continuous line of transformed human embryonic kidney cells, has been recognized for its sensitivity in the isolation of adenoviruses, particularly the fastidious species 40 and 41, from stool specimens. To explore the possibility of using this cell line for the isolation of other viruses from clinical specimens, 293 cells were tested for their susceptibility to a variety of viruses including herpes simplex virus, parainfluenza viruses, respiratory syncytial virus, and the enteroviruses ECHO 11, coxsackie B5, and coxsackie B6. All of the viruses induced a cytopathic effect in 293 cells. Consequently, 293 cells were introduced into the diagnostic laboratory and used along with primary African green monkey kidney (AGMK) cell cultures for the inoculation of all respiratory and stool specimens. The study represents a retrospective analysis of the performance of 293 cells over a 22-month period. It was confirmed that 293 cells were more sensitive than AGMK cells for the isolation of adenoviruses from both respiratory and stool specimens. The 293 cells were also sensitive for the isolation of enteroviruses (untyped) but more so from stool specimens than from respiratory specimens. Parainfluenza virus and respiratory syncytial virus were only rarely isolated in 293 cells. Herpesvirus isolates were obtained with equal frequency in both 293 and AGMK cells. This retrospective analysis confirms the value of 293 cells for the isolation of adenoviruses and demonstrates that 293 cells are also useful for the isolation of certain enteroviruses from both respiratory and stool specimens.     

Replication of minute virus of mice DNA in adenovirus-infected or adenovirus-transformed cells

The effect of adenovirus infection or transformation on the DNA replication of Minute Virus of Mice (MVM) was studied in human fibroblast cell lines. In WI38, HeLa, and 293 cells MVM infection allowed production of viral NS-1 and capsid proteins with or without adenovirus 2 (Ad2) co-infection. However, MVM DNA replication varied markedly. In HeLa cells MVM DNA was replicated weakly in host nucleoli, and replication was increased markedly by Ad2 co-infection as well as recompartmentalized to Ad2 replication factories. In Ad-transformed 293 cells MVM DNA was replicated very efficiently when infected alone or with Ad2 co-infection although recompartmentalization from nucleoli to replication factories was also seen. In WI38 cells MVM DNA was not replicated under any conditions. The variation in DNA replication in WI38, HeLa, and 293 cells despite viral protein production in all cases suggests that MVM DNA replication is uncoupled from viral gene expression and that host factors required for MVM DNA replication are induced or recompartmentalized by adenovirus infection or transformation.     

Complementation of enteric adenovirus type 40 for lytic growth in tissue culture by E1B 55K function of adenovirus types 5 and 12

The enteric adenovirus type 40 strain Dugan (Ad40) cannot be passaged in HeLa cells, but will grow in 293 cells, which express Ad5 E1 functions. To determine the reason for this limited host range, KB cell lines expressing Ad2 E1A, E1B, or E1A + E1B (L. E. Babiss, C. S. H. Young, P. B. Fisher, and H. S. Ginsberg, 1983, J. Virol. 46, 454-465) have been tested for their ability to support Ad40 replication. Only cell lines which supply E1B functions, but not those expressing E1A alone, are permissive for Ad40, suggesting that Ad40 may require some function supplied by E1B or induced in E1B-containing cells. In coinfection assays Ad40 complements Ad5 dl312 (delta E1A) but not Ad5 dl313 (delta E1B) and is itself complemented by dl312 but not by dl313. Mutants of Ad2 and Ad12 with lesions in E1B 55K or 19K protein have been used to further delineate the requirements for Ad40 growth in HeLa cells. For mutants lacking 55K function there is minimal complementation in either direction, whereas those lacking only the 19K product are able to complement Ad40.     

Isolation and Identification of Enteric Adenoviruses

Thirty-four out of 64 faecal samples with adenovirus particles, as seen by electron microscopy, were found to contain adenovirus 40 or 41 by direct isolation and neutralization in Chang's conjunctival cells, mostly within one week. (Ad40 and 41 candidate viruses are serologically related.) 6 other adenovirus specics were isolated; 6 samples gave equivocal results, and 18 were negative. A genus-specific ELISA with an antihexon coat yielded positive results in 40 out of 55 samples; the test failed to identify adenovirus antigen in 10 out of 17 specimens, which were found negative by culture. All of them were negative by immunfluorescence of inoculated Chang cell cultures. Hence the failures are probably due to insufficient amount of virus in the samples. The predominance of only two adenovirus species associated with gastroenteritis in children and the ease of cultivating and identifying them should help to elucidate their etiological significance.