Diagnosis of Fastidious Enteric Adenoviruses 40 and 41 in Stool Specimens

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1 JOURNAL OF CLINICAL MICROBIOLOGY. Sept p /84/ $02.00/0 Copyright g American Society for Microbiology Vol. 2(0. No. 3 Diagnosis of Fastidious Enteric Adenoviruses 40 and 41 in Stool Specimens MARTHA BROWN,* MARTIN PETRIC, AND PETER J. MIDDLETON Departnent of Virology, The Hospital for Sic k Children, Tor-ontto, Ontar io, Cacnadla M5G IX8 Received 28 March 1984/Accepted 5 June 1984 Thirty-five stool specimens, collected over a 14-week period from pediatric gastroenteritis patients and shown to contain adenovirus by electron microscopy, were inoculated onto 293 and HeLa cells. Virus isolates were characterized by serum neutralization and restriction endonuclease cleavage analysis of viral DNA from infected cells. Adenovirus was isolated upon primary inoculation of 293 cells from all 35 specimens shown to contain adenovirus by electron microscopy. Fastidious adenoviruses 40 and 41 (Ad4O and Ad4l) were found in 17 (49%) of the stool specimens, and 4 of these specimens contained a conventional species (Adl, Adl, Adl8, Ad3l) as well as Ad4O. This was first manifest by the observation that four of the isolates which initially grew only in 293 cells acquired the capacity to grow in HeLa cells upon subsequent passage. In each case, the conventional species was undetectable by DNA analysis in the original inoculum but was selected in 293 cells and became the only one detectable by the second passage. Four other specimens, containing Adl or Ad3l alone, failed to grow initially in HeLa cells but did grow in 293 cells. The results of this study demonstrate therefore that (i) 293 cells are more sensitive than HeLa cells for the isolation of conventional as well as fastidious enteric adenovirus species and (ii) identification of viruses from patient specimens should involve minimal passage of the virus in cell culture, as a single passage can result in misdiagnosis of the virus associated with the infection. Adenoviruses 40 and 41 (Ad40 and Ad4l) are referred to as fastidious enteric adenoviruses because they can be readily detected by direct electron microscopic (EM) examination of stool specimens from patients with gastroenteritis but are refractory to isolation in cell cultures currently used for the propagation of adenoviruses (KB, HeLa, HEp-2, human amnion, human embryonic kidney). However, these viruses do replicate and produce infectious progeny in 293 cells (15), a continuous line of human embryonic kidney cells transformed with AdS DNA (6). Before the use of 293 cells for virus isolation from stool specimens, adenovirus was isolated in this laboratory from less than 50% of the specimens positive for adenovirus by EM (13, 14). A study was then initiated to answer two questions: (i) whether the isolation rate from stool specimens positive for adenovirus by EM could be increased by using 293 cells and (ii) whether Ad4O and Ad4l could be easily distinguished from conventional adenovirus species by their ability to grow in 293 but not in HeLa cells. MATERIALS AND METHODS Collection of specimens. The stool specimens used in this study were among those submitted to the virology laboratory at The Hospital for Sick Children for routine diagnosis from early March to mid-june All specimens selected for the study were from children with diarrhea and were shown to contain adenovirus by direct EM examination. With one exception, the age of the patients ranged from 2 weeks to 2 years, with a mean age of 7 months. One of the Ad4O isolates was obtained from a 7-year-old boy with an ileostomy. Cells and virus. The 293 cells were obtained from F. Graham, McMaster University, at passage 43 and were used in this study between passages 60 and 80. The cells were grown in minimal essential medium (Auto-Pow; Flow Laboratories, Inc.) supplemented with 10% fetal bovine serum, penicillin (300 [Lg/ml), and streptomycin (250 plg/ml). Cells * Corresponding author. 334 were subcultured with a trypsin-edta solution containing 500 [ig of trypsin per ml (1:250). 140 mm NaCI, 5.4 mm KCI, 6 mm glucose, 6.9 mm NaHCO mm EDTA, and % phenol red. For the primary isolation of virus from stool specimens, cells were seeded in 24-well tissue culture plates so that the cells were subconfluent at the time of inoculation. within 2 to 3 days after being seeded. Cells infected for the purpose of extracting viral DNA for restriction endonuclease analysis were seeded in 60-mm petri plates and infected the day after seeding. Stool specimens were suspended in a minimal volume of minimal essential medium with a Vortex mixer and clarified by centrifugation in an Eppendorf centrifuge at 12,000 x g for 5 min. For routine isolation, cells were inoculated with a 1/10 dilution of the supernatant fluid. The type strains of Ad4O (Dugan) and Ad4l (Tak) were obtained from the American Type Culture Collection, and stocks were prepared in 293 cells. Preparation of viral DNA. Virus preparations (clinical specimens and prototype strains) were inoculated onto 1- day-old subconfluent monolayers of 293 cells in 60-mm petri dishes. When the cytopathic effect (CPE) was well developed, cells were scraped into the culture fluid with a rubber policeman, transferred to a centrifuge tube, and pelleted at 75 x g for 5 min. The cell pellet was suspended in 0.3 ml of 20 mm Tris (ph 7.5)-10 mm EDTA and transferred to a siliconized microcentrifuge tube (1.5-ml capacity). Viral DNA was isolated by a modification of the Hirt procedure (7) as follows. Sodium dodecyl sulfate was added to a concentration of 0.6% along with proteinase K (Boehringer- Mannheim Biochemicals) (final concentration, 250 p.g/ml). After incubation for 1 h at 37 C, 5 M NaCI was added to a final concentration of 1 M, and the preparation was held at 4 C overnight. The microcentrifuge tube was placed in an International ultracentrifuge SB283 bucket and spun at 17,000 x g for 30 min to pellet the high-molecular-weight cellular DNA. The supernatant fluid was placed into a

2 VOL. 20, 1984 DIAGNOSIS OF Ad40 AND Ad4l 335 second siliconized microcentrifuge tube and extracted once with phenol saturated with Tris-hydrochloride (ph 8.1) and once with chloroform-isoamyl alcohol (24:1). After precipitation with ethanol for 1 h at -70 C, the DNA was collected by centrifugation at 12,000 x g in an Eppendorf microcentrifuge. The pellet was suspended in 10 mm Tris-hydrochloride (ph 7.4)-10 mm MgClI and digested with RNase (25 pg/ml) for 1 h at 37 C, and the DNA was precipitated with ethanol. After drying, the DNA pellet was suspended in 10 mm Trishydrochloride (ph 7.4) and stored at -20 C. Restriction enzyme digestion. Restriction enzymes HinzdIII and SmnaI were purchased from Boehringer-Mannheim. The digestion conditions were those described by Bethesda Research Laboratories, Inc. An HindIII digest of X DNA was purchased from Bethesda Research Laboratories. Gel electrophoresis. Samples were electrophoresed through 5% polyacrylamide gels (polyacrylamide-bisacrylamide, 30:0.8) in 0.5x TBE (ph 8.3) (1x TBE is 89 mm Tris plus 90 mm boric acid plus 2.5 mm disodium EDTA). Electrophoresis was at constant voltage for ca. 1,000 V-h. Staining of gels. Polyacrylamide gels were stained with silver, using the photochemical method described by Beidler et al. (1). Neutralization tests. The adenovirus isolates were classified according to species in microneutralization tests with 293 cells. Specific antisera against Adl through Ad7, Ad8 through Ad3l. and Ad32 through Ad39 were obtained from Microbiological Associates, the National Institutes of Health, and the Centers for Disease Control, respectively. Antisera against Ad4O and Ad4l were a generous gift from G. Wadell, Umea, Sweden. RESULTS Classification of the basis of CPE. Of the 35 specimens examined, all induced CPE in 293 cells (293+), whereas only 17 induced CPE in HeLa cells (HeLa+) upon primary inoculation (Fig. 1). Progeny virus from the cells in which CPE was induced was then passaged as follows. Cells were suspended in the culture fluid and transferred to a tube, and the virus was released by freezing and thawing. The suspension was clatified by centrifugation at 75 x g for 10 min and used to inoculate fresh 293 and HeLa cells. Four distinct growth patterns in 293 and HeLa cells were 17 HeLa specimens recognized. Of the 17 specimens which were HeLa+ upon primary inoculation, 14 were successfully passaged to HeLa cells (group I), whereas 3 were not (group Il). Two of these three specimens did, however, initiate infection when passaged from HeLa to 293 cells. Of the 18 specimens which induced CPE only in 293 cells and not in HeLa cells, i.e., the prospective fastidious enteric adenoviruses, 11 could not be passaged from 293 cells to HeLa cells (group III), whereas 7 which were 293t HeLa- on primary inoculation did induce CPE in HeLa cells after passage through 293 cells (group IV) Ȯf these four groups, it appeared that group I contained conventional adenovirus species with the ability to grow in HeLa cells upon primary inoculation. Group III appeared to represent the fastidious enteric adenoviruses, which grew only in 293 cells and not in HeLa cells. The identities of group II specimens, i.e., those which induced CPE in HeLa cells only upon primary inoculation but could not be passaged to fresh HeLa cells, and group IV specimens, i.e., those which grew on HeLa cells only after passage through 293 cells, were not clear. The virus isolates in each group were therefore characterized by serum neutralization tests and DNA restriction analysis. Neutralization tests. The isolates in group I were classified according to species by microneutralization tests in 293 cells. Altogether, 1 isolate was Ad2, 2 were Ad5, one was Ad18. and 10 were Ad3l. The group IV specimens were also tested by microneutralization after serial passage in 293 cells. Three isolates were Adl, one was Adl8, and three were Ad3l. DNA restriction analysis. The isolates in groups II, III, and IV were characterized by DNA restriction analysis. The three isolates in group II were fastidious enteric adenoviruses, identified as Ad4O by their Sinail cleavage patterns (Fig. 2). By using SinatI, 10 of the 11 specimens in group III were also identified as fastidious enteric adenoviruses: 2 as Ad4l and 8 as Ad4O (Fig. 2). One specimen (Fig. 2) was neither Ad4O nor Ad4l and was identified by neutralization as Ad3l. The Ad4O and Ad4l isolates were neutralized by specific antisera In microneutralization tests. The group IV isolates were examined in an effort to account for their acquired ability to grow in HeLa cells. Duplicate plates were inoculated with the original stool 1 8 HeLa HeLa + HeLa- HeLa- HeLa+ HeLa HeLa- HeLa Ad31(10) Ad 40 (3) Ad 2 (1) Ad 5 (2) Ad 18 (1) l li III Ad 40 (8) Ad41 (2) Ad31 (1) IV Adl (1) Ad 31 (2) Ad 40/1 (2) Ad 40/18 (1) Ad 40/31 (1 ) FIG. 1. Identification of adenovirus isolates from stool specimens shown to be positive for adenovirus by EM. 293 and HeLa cells were incubated with clarified stool suspensions diluted 1/10 and monitored for development of CPE (I+) or absence of CPE (-). Progeny virus from HeLa+ and 293+ cells was passaged to both HeLa and 293 cells. Blind passages from HeLa cells were not done. The isolates within each group are listed. The number of isolates is shown in parentheses.

3 336 BROWN, PETRIC, AND MIDDLETON Hind III Sma '' IM S'E> SI; H 9i j~~~~~~~~~~~~- FIG. 2. DNA restriction patterns of representative isolates from groups and The numbers above each track denote the adenovirus species analyzed. 40+ and 411 refer to the prototype strains of Ad4O (Dugan) and Ad4l (Tak), respectively. The other isolates, including the single Ad3l isolate from group A; 4 4 were from patient specimens. Minor bands (0) in two of the Ad4O isolates suspension and with serially passaged material, i.e., progeny from HeLaTh cells. DNA restriction patterns were then compared. Two of the specimens were identified as Ad3l and one as Adl from the original stool suspension, using as standards the restriction patterns of the group IV isolates identified by neutralization. The remaining four specimens were identified as Ad4O in the original stool specimen but were identified as conventional species (Adl, Adl, Ad18, Ad3l) in the passaged material. Therefore, the acquired ability of these specimens to grow in HeLa cells after ivx~~~~~~~~- passage through 293 cells was accounted for by (i) amplification of a conventional species (Adl, Ad3l, Ad3l), presumably at low infectious titer in the stool, in three cases and bu amplification of a conventional species (Adl, Adl, Adl8, Ad31), present along with an excess of Ad4O, in four cases. The conventional species was selected over Ad4O during passage through 293 cells. DISCUSSION Thirty-five stool specimens which were positive for adenovirus by EM were classified into two groups on the basis of CPE upon primary inoculation of HeLa and 293 cells. Only half of the specimens induced a CPE in HeLa cells, consistent with previous results from this laboratory (13, 14), whereas all of the specimens initiated a productive infection in 293 cells. J. CLIN. MICROBIOL. It was initially thought that it might be possible to distinguish fastidious enteric adenoviruses from conventional species on the basis of their growth patterns in 293 and HeLa cells. However, this proved not to be the case. Based on the absence of CPE in HeLa cells, 18 of the specimens would have been classified as fastidious enteric adenoviruses. In fact, three of these were subsequently identified as Ad3l and one as Adl. Moreover, three specimens which induced a CPE upon primary inoculation of HeLa cells and thus would have been classified as conventional species were identified by restriction analysis as Ad4O (group II). The fact that virus was passaged from HeLa' cells to 293 cells but not to HeLa cells in two of these cases may reflect the high concentration of virus in the original specimen, so that a sufficient amount remained to initiate infection in 293 cells after "passage" through HeLa cells. Alternatively, HeLa cells infected at a high multiplicity of infection may release a small amount of infectious progeny sufficient to initiate productive infection in the sensitive 293 cells but not in HeLa cells. In a previous study, Kidd and Madeley (12) reported the induction of CPE in human embryonic kidney cells by some Ad4O/41 (not differentiated) specimens, but the virus could not be passaged. The results of Kidd and Madeley (12), along with those reported here, are consistent with immunofluorescence studies which demonstrated that human embryonic kidney, human epithelial (HEp-2), and human amnion cells undergo abortive infection by Ad4O/41 (5, 14). In this study, it is unusual that an isolate of Ad3l was included in group III. Even after two passages in 293 cells, it did not induce CPE in HeLa cells. These results thus illustrate the value of 293 cells for the isolation of both conventional and fastidious species of adenovirus. Of particular interest were the seven specimens (group IV) which appeared to be fastidious enteric adenoviruses on the basis of their failure to induce CPE on primary inoculation of HeLa cells but which acquired the ability to grow in HeLa cells after passage through 293 cells. Two of these were identified as Ad3l and one as Adl in the original stool specimens. This would suggest that the infectious titer of virus in the original specimen was low, even though the number of particles was sufficient to be seen by EM and required amplification in 293 cells to cause obvious CPE in HeLa cells. The other four specimens in group IV contained a conventional species (Adl, Adl, Adl8, Ad3l) in addition to Ad4O. It is not uncommon to find more than one species in stool specimens, since conventional species can be shed in the stool for extended periods after respiratory or enteric infections (4, 11). In this case, Ad4O was the only virus detectable by restriction analysis of DNA from high-salt extracts of cells inoculated with the original stool specimen (Fig. 3), thus indicating that Ad4O was associated with the current episode of disease. After a single passage in 293 cells, however, the conventional species was the only one detectable (Fig. 3). Thus, identification of the isolate from the "passage 2" material, as is often done in neutralization tests, would have resulted in misdiagnosis of the virus associated with the disease. It is interesting that the conventional species was selected over Ad4O in 293 cells, which are normally permissive for Ad4O. Moreover, the selection was evident within a single passage. This may simply reflect a faster growth rate of the conventional species relative to Ad4O, or it may be related in some way to the host-range restriction exhibited in culture by Ad4O. Experiments are currently in progress to examine these two possibilities. Within group I, Ad3l was the predominant species (10 out of 14 isolates). This identifies Ad3l as the predominant strain

4 VOL. 20, 1984 /\ /\ / FIG. 3. SmnaI cleavage patterns of DNA from isolates in group IV. The numbers immediately above each track denote the adenovirus species analyzed. The numbers in the upper line denote the passage level of virus used to infect the cells, with zeros representing cells inoculated with the original stool suspension. In the case of four specimens (A ), cells were infected with stool suspension and with passaged virus from the same specimen. 40' represents the prototype strain of Ad4O (Dugan). Minor bands (0) in two Ad4O isolates represent partial digestion fragments. of adenovirus circulating in the population during the 14- week period over which these specimens were collected. In previous reports, the relative incidence of Ad4O and Ad4l in pediatric gastroenteritis has been approximately equal (3, 16). It was unexpected, therefore, that only 2 of the fastidious enteric strains isolated were Ad4l, whereas 15 were Ad4O. This may be due to the small sample size in this study and indicates that Ad4O and Ad3l were the predominant strains circulating in the population over the fairly short time period studied. Other laboratories have reported difficulty in growing Ad4, even in 293 cells (2, 16). No such difficulties were encountered in this study. it is noteworthy that the circulating strains of Ad4O and Ad4l in our population are variants of the prototype strains Dugan (Ad4O) and Tak (Ad4l), isolated in Holland in 1973 and 1979, respectively (3). Although our Ad4O isolates had SaIGa cleavage patterns identical to that of Ad4O (Dugan), they were distinguished as variants of the prototype strain by cleavage with Hindlll. The two Ad4l isolates were identified as variants of the prototype strain Tak by their SinaI as well as their Hindill cleavage patterns. Kidd et al. (10) recently isolated a variant of Ad4l from a patient in Scotland and have labeled the variant Ad4la. DIAGNOSIS OF Ad40 AND Ad4l 337 This study demonstrated that fastidious enteric adenoviruses could not be identified simply by their differential growth patterns in 293 and HeLa cells, since Ad4O at high concentration induced CPE in HeLa cells, and conventional species at low concentration induced CPE in 293 but not HeLa cells. A previous report on the identification of fastidious enteric adenoviruses from stool specimens relied on neutralization of the virus with species-specific (typespecific) antiserum after isolation and serial passage in susceptible cells (11). However, passage of the virus beyond primary isolation in cell culture can lead to erroneous results, as seen in this study with 4 of 17 specimens containing Ad4O or Ad4l. A recent study by Wigand et al. (17) described a method for the concurrent isolation and identification of Ad4O/41 from stool specimens, based on neutralization of CPE. This involved inoculation of Chang conjunctiva and HeLa cells with stool suspensions both untreated and treated with antiserum prepared against purified Ad4O virions. Ad4O and Ad4l cross-react serologically and thus were not distinguished from each other. Moreover, specimens containing a conventional adenovirus species, in addition to Ad4O/41, would not be completely neutralized and hence not identified as Ad4O/41. Given that approximately half of pediatric adenovirus gastroenteritis cases are associated with Ad4O or Ad41 (16-18), there is a need for the identification of Ad4O/41 directly from stool specimens. Chiba et al. (2) used DNA hybridization to identify Ad4O directly from stool specimens which were positive for adenovirus by EM. However, it is not clear whether this involved purification of virions from the stool before extraction of the DNA or whether viral DNA was extracted directly from the stool specimen. We have attempted to purify viral DNA directly from the stool specimens for restriction analysis. This was successful in only 5 of 15 cases (data not shown) and, therefore, was not reproducible. Uhnoo et al. (16) refer to an enzyme immunoassay for direct identification of Ad4O and Ad41 from stool specimens. This involves the use of polyclonal antisera which have been rendered species specific (type specific) by affinity chromatography (8, 9). Such an assay deserves attention. Used with species-specific monoclonal antibodies, it has the potential to simplify and standardize the specific diagnosis of Ad4O and Ad41. Until such antibodies are widely available, the definitive identification of Ad4O and Ad4l will continue to rest on the restriction analysis of viral DNA after isolation of the virus in culture. ACKNOWLEDGMENTS This work was supported by grant MA6401 from the Medical Research Council of Canada and by funds obtained from The Hospital for Sick Children Research Institute. M.B. is a Scholar of the Medical Research Council of Canada. We acknowledge the technical assistance of Antonietta Cifelli. LITERATURE CITED 1. Beidler, J. L., P. R. Hilliard, and R. L. Rill Ultrasensitive staining of nucleic acids with silver. Anal. Biochem. 126: Chiba, S., I. Nakamura, S. Urasawa, S. Nakata, K. Taniguchi, K. Fujinaga, and T. Nakao Outbreak of infantile gastroenteritis due to type 40 adenovirus. Lancet ii: de Jong, J. C., R. Wigand, A. H. Kidd, G. Wadell, J. G. Kapsenberg, C. J. Muzerie, A. G. Wermenbol, and R. G. Firtzlaff Candidate adenoviruses 40 and 41: fastidious adenoviruses from human infant stool. J. Med. Virol. 11: Fox, J. P., C. D. Brandt, F. E. Wasserman, C. E. Hall, S.

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