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A simple immunoperoxidase method for detecting enteric adenovirus and rotavirus in cell culture
ffournal of Infection (1984) 8, 22-27
A s i m p l e i m m u n o p e r o x i d a s e m e t h o d for d e t e c t i n g enteric a d e n o v i r u s and rotavirus in cell c u l t u r e Roberto Cevenini, Fabio Rumpianesi, Raffaella Mazzaracchio, Manuela Donati, Elisabetta Falcieri* and Israel Sarovt
Institute of Microbiology and *Institute of Electron Microscopy, University of Bologna School of Medicine, Ospedale S. Orsola, 9 Via Massarenti, 40138 Bologna, Italy tDepartment of Virology, Faculty of Health Sciences, Ben Gurion University of the Negev, Beer Sheva, Israel Accepted for publication 23 May 1983 Summary A technique which includes the use of indirect immunoperoxidase antibody (IPA) has been developed for detecting enteric adenovirus and rotavirus antigens in cell cultures and has been compared with immunofluorescence antibody assay (IFA). The IPA technique was as sensitive as the IFA. The number of positive cells detected by both techniques in tissue cultures was the same; false positive results were not observed. The applicability of IPA in clinical virology is discussed.
Introduction In the past decade, rotaviruses have been shown to be the major cause of gastroenteritis in infants. 1 More recently, it has been claimed that enteric adenoviruses may also be of aetiological relevance in this disease 2 and cause some 5 to I5 per cent of cases of infantile gastroenteritis in many parts of the world. Several techniques have been described for detecting rotavirus or rotavirus antigens in faeces. Adenovirus antigens also have been detected in faeces by radioimmunoassay and enzyme-linked immunosorbent assay (ELISA). 3 Enteric adenoviruses, which have been observed by electron microscopy in faecal samples of young children with diarrhoea, are often fastidious agents that fail to propagate efficiently and to induce a cytopathic effect in human cell lines.4 Some enteric adenoviruses have recently been reported to induce cytopathic changes in Chang conjunctival cells, 5 and in 293 cells ~ (human embryonic kidney cells transformed by adenovirus type 5)- Notwithstanding the difficulties in growing enteric adenovirus, viral antigens can be detected by immunofluorescence in primary or continuous human cell lines infected with enteric adenovirus. 7 In the present study we have developed a simple immunoperoxidase technique for detecting rotavirus and enteric adenovirus antigens in cell culture and have compared it with an immunofluorescence technique.
Correspondence and reprint requests: Dr Roberto Cevenini.
o163-4453/84/OlOO21+09 $o2.oo/o
©1984 The British Society for the Study of Infection
Adenovirus and rotavirus detection
23
Materials and m e t h o d s Specimens
Thirty faecal samples from infants with acute diarrhoea, positive for rotavirus by electron microscopy, E L I S A and immunofluorescence (as already reported by us), 8 were used in this study. Twenty-three stools from infants with acute diarrhoea, positive for adenovirus by electron microscopy, were studied also. These specimens were part of a study of the aetiology of acute diarrhoea in 449 young children (6 to 24 months of age) begun in I982 and still in progress. P r e p a r a t i o n of viral extracts
Viral extracts were prepared from faecal samples as follows. A 2o per cent suspension of faeces was made in phosphate buffered saline (PBS). T h e suspension was centrifuged at I2oo g for 15 minutes at 4 °C. T h e supernatants were used in subsequent experiments. Electron m i c r o s c o p y
This was performed according to the technique of Flewett and colleaguesfl as previously described. 8 Briefly, the faecal extracts in PBS were concentrated by ammonium sulphate precipitation, applied to a carbon coated 4oo mesh E M grid and negatively stained with phosphotungstic acid (pH 6.6). Cell cultures for rotavirus and adenovirus
Monolayers of monkey kidney cells of L L C - M K 2 line were used for detecting rotavirus antigens, by means of the technique of Bryden and colleagues 1° as previously described. 8 Briefly, coverslips with confluent monolayers of L L C - M K 2 cells were infected with 30 ~tl of faecal extracts and centrifuged at 1200 g for I hour. After incubating them for 24 hours at 37 °C, the coverslip cultures were fixed in acetone, air dried, and used as antigen. H u m a n embryo fibroblast (HEF) and human epidermoid carcinoma (HEp-2) cells grown on 12 m m round coverslips at the bottom of a Linbro Tissue culture multi-weU plate (Flow Laboratories Inc, U.S.A.) were inoculated with o.2 ml faecal extracts for adenovirus detection. After 3 and 7 days' incubation respectively at 37 °C in 5 per cent carbon dioxide (CO2) two coverslips of infected H E F and HEp-2 cells were fixed in cold methanol for 5 minutes and used as antigen in IFA and IPA assays. In addition, after 7 days of incubation, infected H E F and HEp-2 cell cultures were harvested by freezing and thawing twice, clarified by centrifuging at IOOO g for 15 minutes and then subpassaged at least four times. At each subpassage, two coverslips of H E F and two of HEp-2 cells were fixed in methanol 3 and 7 days after infection. I m m u n o f l u o r e s c e n c e assays
Rotavirus T h e fixed L L C - M K 2 cells were treated, as previously described, s for 3o minutes at 37 °C with a I in IO dilution of hyperimmune bovine rotavirus antiserum (purchased from Professor U. Krech, St Gallen, Switzerland) washed in PBS and stained for 3o minutes with fluorescein-conjugated rabbit
24
R. CEVENINI E T A L .
anti-bovine serum (Wellcome Research Laboratories, Beckenham, England) then washed in PBS, counterstained with Evans blue o.oi per cent in distilled water, and finally m o u n t e d in buffered glycerol p H 8"5. Enteric adenovirus T h e fixed H E F and HEp-2 cell cultures were treated as described for rotavirus, except for the i m m u n e serum which was a rabbit adeno anti-hexon i m m u n e serum (purchased from Professor U. Krech, St Gallen, Switzerland). Antirabbit i m m u n e globulin (sheep) fluorescein-labelled (Wellcome Reagents) diluted I in 4o was used as the second antibody. Slides were observed by means of a Zeiss-UV microscope. Immunoperoxidase assays
T h e fixed slides were treated for 3o minutes at 37 °C with a I in IO dilution of the h y p e r i m m u n e bovine rotavirus serum or with the rabbit andeno anti-hexon i m m u n e serum. After incubation, the slides were washed for 15 minutes in PBS and incubated for 3o minutes with anti-bovine Ig peroxidase conjugate (Kirkegaard and Perry Laboratories, Gaithersburg, M d , U.S.A.) diluted I in 3o in PBS, or with anti-rabbit Ig peroxidase conjugate (Dako, Copenhagen, Denmark) diluted i in 3o in PBS, for rotavirus or adenovirus antigen detection, respectively. After washing, the enzymatic activity was detected by means of a modification of the m e t h o d of G r a h a m and Karnowski. 11 T h e freshly prepared substrate solution was made of 4 m g benzidine (Fluka AG, Buchs, Switzerland), dissolved in 0"5 ml acetone, 9"5 ml PBS and IO pl hydrogen peroxide from 33 per cent stock solution. T h e substrate was added for 5 minutes at room temperature and was followed by washing in PBS. Results
Plate I shows the typical dark blue cytoplasmic staining ofrotavirus L L C - M K 2 infected cells, obtained by the peroxidase reaction. T h e staining was always cytoplasmic, sometimes granular in appearance, and without nuclear involvement. Immunoperoxidase and immunofluorescence reactions were entirely similar in their sensitivity for detecting rotavirus. Of the 3o faecal samples positive for rotavirus by electron microscopy, 28 proved to be positive by I P A and IFA. Of the 449 consecutive faecal samples examined for adenovirus by electron microscopy, 23 (5" I per cent) were positive. Of these samples 18 (78"2 per cent) caused a clearly visible cytopathic effect in H E p 2 cells, generally by the third or fourth passage, whereas only two of the specimens caused a cytopathic effect in H E R T h e s e two adenovirus strains were typed by rabbit neutralising antiserum, as adenovirus type I. Five samples failed to produce a marked cytopathic effect even in H E p - 2 cells. Nevertheless, all 23 faecal samples induced the production of adenovirus antigens in H E p - 2 cells, always detectable either by I F A or IPA, 3 or 7 days after infection. On the contrary, adenovirus antigens were sparingly detectable in very few h u m a n embryo fibroblasts, in 15 of the 23 samples, including those from which adenovirus type I was isolated. T h e general pattern of reaction by specific adenovirus antiserum was similar
Journal of Infection
Plate I
!!i~iiiii!i!!!!~
Plate I. Typical dark blue cytoplasmic staining of rotavirus infected L L C - M K 2 cell given by (a) the peroxidase reaction, (b) uninfected LLC--MK2 cells.
CEVENINI ET AL.
(Facing p. 24)
Journal of Infection
Plate 2
Plate 2. HEp-2 cells infected with an enteric adenovirus and stained by the peroxidase reaction. (a) infected cells with crystals on the cell surface, (b) a typical group of a few infected cells showing the nuclear and cytoplasmic staining given by the peroxidase technique, (c) uninfected HEp-2 cells. CEVENINI E T
AL.
Journal of Infection
Plate 3
Plate 3. HEp-2 cells inoculated with an enteric adenovirus and stained by the immunofluorescence antibody technique.
CEVENINI E T
AL.
Adenovirus and rotavirus detection
25
with both techniques. T h e typical staining given by the peroxidase technique was observed both in the nuclei and in the cytoplasm of the infected cells. T h e staining intensity changed from a dark blue to a pale brown. Rarely the staining was granular and often crystals were present at the cell surface (Plate 2). T h e infected cells stained by IFA had a similar appearance: there was strong fluorescence in the nuclei and usually staining of the cytoplasm (Plate 3). T h e staining observed at the first passage either by IPA and IFA was mostly confined to single cells. Sometimes loci of a few infected cells were also observed. T h e percentage of positive cells detected in tissue culture samples was identical by both techniques. T h e number of infected HEp-2 cells increased by subpassaging, at least up to the fourth passage. On the contrary, by subpassaging H E F infected cells (with the exception of adenovirus I infected cells), there was not any increase, but a decrease, in the number of positive cells, both by IPA and IFA. Discussion
Acute viral gastroenteritis is an extremely common illness that affects all age groups, in particular young children. Recently, the availability of advanced laboratory techniques has made it possible to define rotaviruses as the major pathogens in infantile gastroenteritis. Several other agents, including adenoviruses, have also been seen by direct electron microscopy in diarrhoeal faeces and have been proposed as possible causes of viral gastroenteritis. Adenovirus infections are associated with many kinds of illness in human beings. Recently new serotypes have been established: Ad 34 isolated from urine, 1~ Ad 35 from lung and kidney, 13Ad 36 from stools, 14Ad 37 from eye, cervix and urethra, 15,16 Ad 38 which represents the 'non cultivable' or enteric adenovirus isolated from stool specimens, 17 and Ad 39 recently isolated from stools of a patient with respiratory illness. 18 It has been known for many years that adenoviruses can be isolated from stools but some adenoviruses which can be detected by electron microscopy fail to replicate efficiently in any conventional cell line. Such strains, e.g. Ad 38, may be new serotypes or strains of one or more of the 39 established serotypes that are difficult to isolate. Recently, some enteric adenoviruses have been partially or completely adapted to grow in particular cell lines such as Chang conjunctival cells 5 or 293 cells, e In the present study, all 23 adenoviruses detected in stools by electron microscopy were completely or partially adapted to grow in HEp-2 cells, but not in H E F , with the exception of two strains typed as Ad I, which grew well in HEF. Eighteen of the 23 Ad strains produced a marked cytopathic effect in HEp-2 cells, while five produced a rounding of single cells scattered in the cell monolayers. Also in these cases, however, the number of infected cells, as detected by IFA and IPA, progressively increased, at least up to the fourth passage. Studies are still in progress in our laboratory to correctly type these enteric adenoviruses and to further assess the possibility of adapting these five strains to grow in HEp-2 cells. Many techniques have already been reported for detecting rotavirus or enteric adenovirus in stools: electron microscopy, 1, 2.9 E L I SA, 19 radioimmunoassay,3 immunoelectroosmophoresis,2° immunofluorescence in cell culture, 3, 21 including a peroxidase-antiperoxidase technique
26
R. C E V E N I N I E T AL.
f o r d e t e c t i n g S i m i a n r o t a v i r u s in cell c u l t u r e . 2~ R e c e n t l y in o u r l a b o r a t o r y t h e reliability of the indirect immunoperoxidase technique has been evaluated and c o m p a r e d w i t h o t h e r t e c h n i q u e s f o r d e t e c t i n g a n t i b o d y in s e r u m s a m p l e s . 2a-~5 Complete correlation was found between the various methods and IPA. In the present study we developed an indirect immunoperoxidase technique f o r d e t e c t i n g h u m a n r o t a v i r u s a n d e n t e r i c a d e n o v i r u s a n t i g e n s in cell c u l t u r e s . T h e I P A d e s c r i b e d w a s as s e n s i t i v e as t h e I F A t e c h n i q u e since t h e n u m b e r o f p o s i t i v e cells d e t e c t e d b y b o t h t e c h n i q u e s in t h e c u l t u r e s w a s i d e n t i c a l ; false positive or negative results were never observed. Some advantages of immunoperoxidase technique over the immunofluorescence technique are that p e r m a n e n t slides are p r o d u c e d a n d a n e x p e n s i v e f l u o r e s c e n c e m i c r o s c o p e is n o t required. T h i s I P A t e s t a p p l i e d to t h e d e t e c t i o n o f r o t a v i r u s o r e n t e r i c a d e n o v i r u s a n t i g e n s in t i s s u e c u l t u r e s s e e m s to b e s u i t a b l e f o r clinical v i r o l o g y l a b o r a t o r i e s b e c a u s e t h e r e a g e n t s are easily p r e p a r e d a n d t h e r e a d i n g d o e s n o t s e e m to b e difficult. I t m a y offer a n a l t e r n a t i v e to o t h e r m o r e s o p h i s t i c a t e d t e c h n i q u e s , d e p e n d i n g o n facilities a v a i l a b l e . (We thank M r L o r e d a n o Franchi and M r Vittorio Sambri for their excellent technical work.) References
Flewett TH, Bryden AS, Davies H. Virus particles in gastroenteritis. Lancet 1973; 2: I4972. Richmond SJ, Caul EO, Dunn SM, Asheley CR, Clarke SKR, Seymour NR. An outbreak of gastroenteritis in young children caused by adenovirus. Lancet 1979; I: 1178-I I8O. 3. Sarkkinen HK, Tuokko H, Halonen PE. Comparison of enzyme-immunoassay and radioimmunoassay for detection of human rotavimses and adenoviruses from stool specimens. ff Virol Meth 198o; I: 331-34I. 4. Retter M, Middleton PJ, Tam JS, Petric M. Enteric adenoviruses: detection, replication and significance, ff Clin Microbiol 1979; xo: 574-578. 5. Kidd AH, Madeley CR. In vitro growth of some fastidious adenovirus from stool specimens. ff Clin Pathol I98I; 34: 213--2166. Takiff HE, Straus S, Garon CF. Propagation and in vitro studies of previously noncultivable enteral adenovirus in 293 cells. Lancet I98 I; 2: 832-834. 7. Gary GW, Hierholzer JC, Black RE. Characteristics of noncultivable adenovirus associated with diarrhoea in infants: a new subgroup of human adenovirus, ff Clin Microbiol I979; IO: 96-IO3 . 8. Cevenini R, Rumpianesi F, Donati M, Falcieri E, Lazzari R. A comparative study of different techniques for the detection ofrotaviruses in stools. Microbiologica 1982; 5 : 13 z-135. 9. Flewett T H , Davies HA, Bryden AS, Robertson MJ. Diagnostic electron microscopy of faeces. II. Acute gastroenteritis associated with reovirus-like particles, ff Clin Pathol I974; 27: 6o8-6 I4. Io. Bryden AS, Davies HA, Thouless ME, Flewett TH. Diagnosis of rotavirus infection by cell culture. J Med Microbiol I977; xo: I2I-I25. 1 I. Graham R, Karnowsky MJ. The early states of absorption of injected horseradish peroxidase in proximal tubule of mouse kidney: ultrastructural cytochemistry by a new technique. J Histochem Cytochem 1966; 14: 291-3o2. i2. Hierholzer JC, Atuk NO, Gwalmey JM. New human adenovirus isolated from renal transplant recipient: description and characterization of candidate adenovirus type 34. ft. Clin Microbiol I975; l : 366-376. I3. Stalder H, Hierholzer JC, Oxman MN. New human adenovirus (candidate adenovirus type 35) causing fatal disseminated infection in renal transplant recipient, ff Clin Microbiol I977; 6: 257-265. i.
A d e n o v i r u s a n d rotavirus detection
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14. Wigand R, Gelderblom H, WadeU G. New human adenovirus (candidate adenovirus 36), a novel member of subgroup D. Arch Virol I98o; 64: 225-233. I5. de Jong JC, Wigand R, Wadell G e t al. Adenovirus 37: identification and characterization of a medically important new adenovirus type of subgroup D. J Med Virol I981 ; 7 : IO5-I 18. I6. Cevenini R, Donati M, Landini MP, La Placa M. Adenovirus associated with an oculo-genital infection. Microbiologica I979; 2: 425-427. I7. de Jong JC, Wigand R, Kidd AH, Kapsenberg JG, Muzerie CJ, Firtzlaff R. Candidate adenovirus 38, associated with human infantile gastroenteritis and related to 'noncultivable' enteric adenovirus. In: Murphy FA, ed. Proceedings of the Fifth International Congress of Virology, Strasbourg, France. Paris: L'Imprimerie Centrale Commerciale, I98I: I94. i8. Hierholzer JC, Kemp MC, Gary JR, Spencer HC. New human adenovirus associated with respiratory illness: candidate adenovirus type 39. J Clin Pathol I982; x6: I5-2I. I9. Yolken RH, Leister FJ. Evaluation of enzymeimmunoassays for the detection of human rotavirus. J Infect Dis I98I; I44: 379. 20. Jacobsson PA, Johansson ME, Wadell G. Identification of an enteric adenovirus by immunoelectroosmophoresis (IEOP) technique. J Med Virol I979; 3:307-3 I2. 2I. Banatvala JE, Totterdell B, Chrystie IL, Woode GN. In vitro detection of human rotavirus. Lancet I975; 2: 82I. 22. Graham DY, Estes MK. Comparison of methods for immunocytochemical detection of rotavirus infections. Infect Immun I979; 26: 686-689. 23. Haikin H, Leventon-Kriss S, Sarov I. Antibody to Varicella-zoster virus-induced membrane antigen: immunoperoxidase assay with air dried target cells, ft. Infect Dis x979; I4O: 60 I-6o4. 24. Sarov I, Levy E, Aymard M e t al. Detection of virus specific IgA antibodies in serum of kidney transplant patients with recurrent Cytomegalovirus infection by enzymeimmuno and radioimmunoassay techniques. Clin Exp Immunol i982; 48: 321-328. 25. Cevenini R, Rumpianesi F, Donati M, Sarov I. A rapid immunoperoxidase assay for the detection of specific IgG antibodies to Chlamydia trachomatis. J. Clin Pathol r983 ; 36: 353-356.
A s i m p l e i m m u n o p e r o x i d a s e m e t h o d for d e t e c t i n g enteric a d e n o v i r u s and rotavirus in cell c u l t u r e Roberto Cevenini, Fabio Rumpianesi, Raffaella Mazzaracchio, Manuela Donati, Elisabetta Falcieri* and Israel Sarovt
Institute of Microbiology and *Institute of Electron Microscopy, University of Bologna School of Medicine, Ospedale S. Orsola, 9 Via Massarenti, 40138 Bologna, Italy tDepartment of Virology, Faculty of Health Sciences, Ben Gurion University of the Negev, Beer Sheva, Israel Accepted for publication 23 May 1983 Summary A technique which includes the use of indirect immunoperoxidase antibody (IPA) has been developed for detecting enteric adenovirus and rotavirus antigens in cell cultures and has been compared with immunofluorescence antibody assay (IFA). The IPA technique was as sensitive as the IFA. The number of positive cells detected by both techniques in tissue cultures was the same; false positive results were not observed. The applicability of IPA in clinical virology is discussed.
Introduction In the past decade, rotaviruses have been shown to be the major cause of gastroenteritis in infants. 1 More recently, it has been claimed that enteric adenoviruses may also be of aetiological relevance in this disease 2 and cause some 5 to I5 per cent of cases of infantile gastroenteritis in many parts of the world. Several techniques have been described for detecting rotavirus or rotavirus antigens in faeces. Adenovirus antigens also have been detected in faeces by radioimmunoassay and enzyme-linked immunosorbent assay (ELISA). 3 Enteric adenoviruses, which have been observed by electron microscopy in faecal samples of young children with diarrhoea, are often fastidious agents that fail to propagate efficiently and to induce a cytopathic effect in human cell lines.4 Some enteric adenoviruses have recently been reported to induce cytopathic changes in Chang conjunctival cells, 5 and in 293 cells ~ (human embryonic kidney cells transformed by adenovirus type 5)- Notwithstanding the difficulties in growing enteric adenovirus, viral antigens can be detected by immunofluorescence in primary or continuous human cell lines infected with enteric adenovirus. 7 In the present study we have developed a simple immunoperoxidase technique for detecting rotavirus and enteric adenovirus antigens in cell culture and have compared it with an immunofluorescence technique.
Correspondence and reprint requests: Dr Roberto Cevenini.
o163-4453/84/OlOO21+09 $o2.oo/o
©1984 The British Society for the Study of Infection
Adenovirus and rotavirus detection
23
Materials and m e t h o d s Specimens
Thirty faecal samples from infants with acute diarrhoea, positive for rotavirus by electron microscopy, E L I S A and immunofluorescence (as already reported by us), 8 were used in this study. Twenty-three stools from infants with acute diarrhoea, positive for adenovirus by electron microscopy, were studied also. These specimens were part of a study of the aetiology of acute diarrhoea in 449 young children (6 to 24 months of age) begun in I982 and still in progress. P r e p a r a t i o n of viral extracts
Viral extracts were prepared from faecal samples as follows. A 2o per cent suspension of faeces was made in phosphate buffered saline (PBS). T h e suspension was centrifuged at I2oo g for 15 minutes at 4 °C. T h e supernatants were used in subsequent experiments. Electron m i c r o s c o p y
This was performed according to the technique of Flewett and colleaguesfl as previously described. 8 Briefly, the faecal extracts in PBS were concentrated by ammonium sulphate precipitation, applied to a carbon coated 4oo mesh E M grid and negatively stained with phosphotungstic acid (pH 6.6). Cell cultures for rotavirus and adenovirus
Monolayers of monkey kidney cells of L L C - M K 2 line were used for detecting rotavirus antigens, by means of the technique of Bryden and colleagues 1° as previously described. 8 Briefly, coverslips with confluent monolayers of L L C - M K 2 cells were infected with 30 ~tl of faecal extracts and centrifuged at 1200 g for I hour. After incubating them for 24 hours at 37 °C, the coverslip cultures were fixed in acetone, air dried, and used as antigen. H u m a n embryo fibroblast (HEF) and human epidermoid carcinoma (HEp-2) cells grown on 12 m m round coverslips at the bottom of a Linbro Tissue culture multi-weU plate (Flow Laboratories Inc, U.S.A.) were inoculated with o.2 ml faecal extracts for adenovirus detection. After 3 and 7 days' incubation respectively at 37 °C in 5 per cent carbon dioxide (CO2) two coverslips of infected H E F and HEp-2 cells were fixed in cold methanol for 5 minutes and used as antigen in IFA and IPA assays. In addition, after 7 days of incubation, infected H E F and HEp-2 cell cultures were harvested by freezing and thawing twice, clarified by centrifuging at IOOO g for 15 minutes and then subpassaged at least four times. At each subpassage, two coverslips of H E F and two of HEp-2 cells were fixed in methanol 3 and 7 days after infection. I m m u n o f l u o r e s c e n c e assays
Rotavirus T h e fixed L L C - M K 2 cells were treated, as previously described, s for 3o minutes at 37 °C with a I in IO dilution of hyperimmune bovine rotavirus antiserum (purchased from Professor U. Krech, St Gallen, Switzerland) washed in PBS and stained for 3o minutes with fluorescein-conjugated rabbit
24
R. CEVENINI E T A L .
anti-bovine serum (Wellcome Research Laboratories, Beckenham, England) then washed in PBS, counterstained with Evans blue o.oi per cent in distilled water, and finally m o u n t e d in buffered glycerol p H 8"5. Enteric adenovirus T h e fixed H E F and HEp-2 cell cultures were treated as described for rotavirus, except for the i m m u n e serum which was a rabbit adeno anti-hexon i m m u n e serum (purchased from Professor U. Krech, St Gallen, Switzerland). Antirabbit i m m u n e globulin (sheep) fluorescein-labelled (Wellcome Reagents) diluted I in 4o was used as the second antibody. Slides were observed by means of a Zeiss-UV microscope. Immunoperoxidase assays
T h e fixed slides were treated for 3o minutes at 37 °C with a I in IO dilution of the h y p e r i m m u n e bovine rotavirus serum or with the rabbit andeno anti-hexon i m m u n e serum. After incubation, the slides were washed for 15 minutes in PBS and incubated for 3o minutes with anti-bovine Ig peroxidase conjugate (Kirkegaard and Perry Laboratories, Gaithersburg, M d , U.S.A.) diluted I in 3o in PBS, or with anti-rabbit Ig peroxidase conjugate (Dako, Copenhagen, Denmark) diluted i in 3o in PBS, for rotavirus or adenovirus antigen detection, respectively. After washing, the enzymatic activity was detected by means of a modification of the m e t h o d of G r a h a m and Karnowski. 11 T h e freshly prepared substrate solution was made of 4 m g benzidine (Fluka AG, Buchs, Switzerland), dissolved in 0"5 ml acetone, 9"5 ml PBS and IO pl hydrogen peroxide from 33 per cent stock solution. T h e substrate was added for 5 minutes at room temperature and was followed by washing in PBS. Results
Plate I shows the typical dark blue cytoplasmic staining ofrotavirus L L C - M K 2 infected cells, obtained by the peroxidase reaction. T h e staining was always cytoplasmic, sometimes granular in appearance, and without nuclear involvement. Immunoperoxidase and immunofluorescence reactions were entirely similar in their sensitivity for detecting rotavirus. Of the 3o faecal samples positive for rotavirus by electron microscopy, 28 proved to be positive by I P A and IFA. Of the 449 consecutive faecal samples examined for adenovirus by electron microscopy, 23 (5" I per cent) were positive. Of these samples 18 (78"2 per cent) caused a clearly visible cytopathic effect in H E p 2 cells, generally by the third or fourth passage, whereas only two of the specimens caused a cytopathic effect in H E R T h e s e two adenovirus strains were typed by rabbit neutralising antiserum, as adenovirus type I. Five samples failed to produce a marked cytopathic effect even in H E p - 2 cells. Nevertheless, all 23 faecal samples induced the production of adenovirus antigens in H E p - 2 cells, always detectable either by I F A or IPA, 3 or 7 days after infection. On the contrary, adenovirus antigens were sparingly detectable in very few h u m a n embryo fibroblasts, in 15 of the 23 samples, including those from which adenovirus type I was isolated. T h e general pattern of reaction by specific adenovirus antiserum was similar
Journal of Infection
Plate I
!!i~iiiii!i!!!!~
Plate I. Typical dark blue cytoplasmic staining of rotavirus infected L L C - M K 2 cell given by (a) the peroxidase reaction, (b) uninfected LLC--MK2 cells.
CEVENINI ET AL.
(Facing p. 24)
Journal of Infection
Plate 2
Plate 2. HEp-2 cells infected with an enteric adenovirus and stained by the peroxidase reaction. (a) infected cells with crystals on the cell surface, (b) a typical group of a few infected cells showing the nuclear and cytoplasmic staining given by the peroxidase technique, (c) uninfected HEp-2 cells. CEVENINI E T
AL.
Journal of Infection
Plate 3
Plate 3. HEp-2 cells inoculated with an enteric adenovirus and stained by the immunofluorescence antibody technique.
CEVENINI E T
AL.
Adenovirus and rotavirus detection
25
with both techniques. T h e typical staining given by the peroxidase technique was observed both in the nuclei and in the cytoplasm of the infected cells. T h e staining intensity changed from a dark blue to a pale brown. Rarely the staining was granular and often crystals were present at the cell surface (Plate 2). T h e infected cells stained by IFA had a similar appearance: there was strong fluorescence in the nuclei and usually staining of the cytoplasm (Plate 3). T h e staining observed at the first passage either by IPA and IFA was mostly confined to single cells. Sometimes loci of a few infected cells were also observed. T h e percentage of positive cells detected in tissue culture samples was identical by both techniques. T h e number of infected HEp-2 cells increased by subpassaging, at least up to the fourth passage. On the contrary, by subpassaging H E F infected cells (with the exception of adenovirus I infected cells), there was not any increase, but a decrease, in the number of positive cells, both by IPA and IFA. Discussion
Acute viral gastroenteritis is an extremely common illness that affects all age groups, in particular young children. Recently, the availability of advanced laboratory techniques has made it possible to define rotaviruses as the major pathogens in infantile gastroenteritis. Several other agents, including adenoviruses, have also been seen by direct electron microscopy in diarrhoeal faeces and have been proposed as possible causes of viral gastroenteritis. Adenovirus infections are associated with many kinds of illness in human beings. Recently new serotypes have been established: Ad 34 isolated from urine, 1~ Ad 35 from lung and kidney, 13Ad 36 from stools, 14Ad 37 from eye, cervix and urethra, 15,16 Ad 38 which represents the 'non cultivable' or enteric adenovirus isolated from stool specimens, 17 and Ad 39 recently isolated from stools of a patient with respiratory illness. 18 It has been known for many years that adenoviruses can be isolated from stools but some adenoviruses which can be detected by electron microscopy fail to replicate efficiently in any conventional cell line. Such strains, e.g. Ad 38, may be new serotypes or strains of one or more of the 39 established serotypes that are difficult to isolate. Recently, some enteric adenoviruses have been partially or completely adapted to grow in particular cell lines such as Chang conjunctival cells 5 or 293 cells, e In the present study, all 23 adenoviruses detected in stools by electron microscopy were completely or partially adapted to grow in HEp-2 cells, but not in H E F , with the exception of two strains typed as Ad I, which grew well in HEF. Eighteen of the 23 Ad strains produced a marked cytopathic effect in HEp-2 cells, while five produced a rounding of single cells scattered in the cell monolayers. Also in these cases, however, the number of infected cells, as detected by IFA and IPA, progressively increased, at least up to the fourth passage. Studies are still in progress in our laboratory to correctly type these enteric adenoviruses and to further assess the possibility of adapting these five strains to grow in HEp-2 cells. Many techniques have already been reported for detecting rotavirus or enteric adenovirus in stools: electron microscopy, 1, 2.9 E L I SA, 19 radioimmunoassay,3 immunoelectroosmophoresis,2° immunofluorescence in cell culture, 3, 21 including a peroxidase-antiperoxidase technique
26
R. C E V E N I N I E T AL.
f o r d e t e c t i n g S i m i a n r o t a v i r u s in cell c u l t u r e . 2~ R e c e n t l y in o u r l a b o r a t o r y t h e reliability of the indirect immunoperoxidase technique has been evaluated and c o m p a r e d w i t h o t h e r t e c h n i q u e s f o r d e t e c t i n g a n t i b o d y in s e r u m s a m p l e s . 2a-~5 Complete correlation was found between the various methods and IPA. In the present study we developed an indirect immunoperoxidase technique f o r d e t e c t i n g h u m a n r o t a v i r u s a n d e n t e r i c a d e n o v i r u s a n t i g e n s in cell c u l t u r e s . T h e I P A d e s c r i b e d w a s as s e n s i t i v e as t h e I F A t e c h n i q u e since t h e n u m b e r o f p o s i t i v e cells d e t e c t e d b y b o t h t e c h n i q u e s in t h e c u l t u r e s w a s i d e n t i c a l ; false positive or negative results were never observed. Some advantages of immunoperoxidase technique over the immunofluorescence technique are that p e r m a n e n t slides are p r o d u c e d a n d a n e x p e n s i v e f l u o r e s c e n c e m i c r o s c o p e is n o t required. T h i s I P A t e s t a p p l i e d to t h e d e t e c t i o n o f r o t a v i r u s o r e n t e r i c a d e n o v i r u s a n t i g e n s in t i s s u e c u l t u r e s s e e m s to b e s u i t a b l e f o r clinical v i r o l o g y l a b o r a t o r i e s b e c a u s e t h e r e a g e n t s are easily p r e p a r e d a n d t h e r e a d i n g d o e s n o t s e e m to b e difficult. I t m a y offer a n a l t e r n a t i v e to o t h e r m o r e s o p h i s t i c a t e d t e c h n i q u e s , d e p e n d i n g o n facilities a v a i l a b l e . (We thank M r L o r e d a n o Franchi and M r Vittorio Sambri for their excellent technical work.) References
Flewett TH, Bryden AS, Davies H. Virus particles in gastroenteritis. Lancet 1973; 2: I4972. Richmond SJ, Caul EO, Dunn SM, Asheley CR, Clarke SKR, Seymour NR. An outbreak of gastroenteritis in young children caused by adenovirus. Lancet 1979; I: 1178-I I8O. 3. Sarkkinen HK, Tuokko H, Halonen PE. Comparison of enzyme-immunoassay and radioimmunoassay for detection of human rotavimses and adenoviruses from stool specimens. ff Virol Meth 198o; I: 331-34I. 4. Retter M, Middleton PJ, Tam JS, Petric M. Enteric adenoviruses: detection, replication and significance, ff Clin Microbiol 1979; xo: 574-578. 5. Kidd AH, Madeley CR. In vitro growth of some fastidious adenovirus from stool specimens. ff Clin Pathol I98I; 34: 213--2166. Takiff HE, Straus S, Garon CF. Propagation and in vitro studies of previously noncultivable enteral adenovirus in 293 cells. Lancet I98 I; 2: 832-834. 7. Gary GW, Hierholzer JC, Black RE. Characteristics of noncultivable adenovirus associated with diarrhoea in infants: a new subgroup of human adenovirus, ff Clin Microbiol I979; IO: 96-IO3 . 8. Cevenini R, Rumpianesi F, Donati M, Falcieri E, Lazzari R. A comparative study of different techniques for the detection ofrotaviruses in stools. Microbiologica 1982; 5 : 13 z-135. 9. Flewett T H , Davies HA, Bryden AS, Robertson MJ. Diagnostic electron microscopy of faeces. II. Acute gastroenteritis associated with reovirus-like particles, ff Clin Pathol I974; 27: 6o8-6 I4. Io. Bryden AS, Davies HA, Thouless ME, Flewett TH. Diagnosis of rotavirus infection by cell culture. J Med Microbiol I977; xo: I2I-I25. 1 I. Graham R, Karnowsky MJ. The early states of absorption of injected horseradish peroxidase in proximal tubule of mouse kidney: ultrastructural cytochemistry by a new technique. J Histochem Cytochem 1966; 14: 291-3o2. i2. Hierholzer JC, Atuk NO, Gwalmey JM. New human adenovirus isolated from renal transplant recipient: description and characterization of candidate adenovirus type 34. ft. Clin Microbiol I975; l : 366-376. I3. Stalder H, Hierholzer JC, Oxman MN. New human adenovirus (candidate adenovirus type 35) causing fatal disseminated infection in renal transplant recipient, ff Clin Microbiol I977; 6: 257-265. i.
A d e n o v i r u s a n d rotavirus detection
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14. Wigand R, Gelderblom H, WadeU G. New human adenovirus (candidate adenovirus 36), a novel member of subgroup D. Arch Virol I98o; 64: 225-233. I5. de Jong JC, Wigand R, Wadell G e t al. Adenovirus 37: identification and characterization of a medically important new adenovirus type of subgroup D. J Med Virol I981 ; 7 : IO5-I 18. I6. Cevenini R, Donati M, Landini MP, La Placa M. Adenovirus associated with an oculo-genital infection. Microbiologica I979; 2: 425-427. I7. de Jong JC, Wigand R, Kidd AH, Kapsenberg JG, Muzerie CJ, Firtzlaff R. Candidate adenovirus 38, associated with human infantile gastroenteritis and related to 'noncultivable' enteric adenovirus. In: Murphy FA, ed. Proceedings of the Fifth International Congress of Virology, Strasbourg, France. Paris: L'Imprimerie Centrale Commerciale, I98I: I94. i8. Hierholzer JC, Kemp MC, Gary JR, Spencer HC. New human adenovirus associated with respiratory illness: candidate adenovirus type 39. J Clin Pathol I982; x6: I5-2I. I9. Yolken RH, Leister FJ. Evaluation of enzymeimmunoassays for the detection of human rotavirus. J Infect Dis I98I; I44: 379. 20. Jacobsson PA, Johansson ME, Wadell G. Identification of an enteric adenovirus by immunoelectroosmophoresis (IEOP) technique. J Med Virol I979; 3:307-3 I2. 2I. Banatvala JE, Totterdell B, Chrystie IL, Woode GN. In vitro detection of human rotavirus. Lancet I975; 2: 82I. 22. Graham DY, Estes MK. Comparison of methods for immunocytochemical detection of rotavirus infections. Infect Immun I979; 26: 686-689. 23. Haikin H, Leventon-Kriss S, Sarov I. Antibody to Varicella-zoster virus-induced membrane antigen: immunoperoxidase assay with air dried target cells, ft. Infect Dis x979; I4O: 60 I-6o4. 24. Sarov I, Levy E, Aymard M e t al. Detection of virus specific IgA antibodies in serum of kidney transplant patients with recurrent Cytomegalovirus infection by enzymeimmuno and radioimmunoassay techniques. Clin Exp Immunol i982; 48: 321-328. 25. Cevenini R, Rumpianesi F, Donati M, Sarov I. A rapid immunoperoxidase assay for the detection of specific IgG antibodies to Chlamydia trachomatis. J. Clin Pathol r983 ; 36: 353-356.