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A simple and rapid method for typing adenoviruses 40 and 41 without cultivation
joar~ai of Viro~ogica~ ~ethads,
39
10 (1985) 39-44
Elsevier JVM 00356
A SIMPLE AND RAPID METHOD WITHOUT
JOHANNES
FOR TYPING ADENOVIRUSES
CULTIVATION
BUITENWERF’,
J.J. LOUWERENS’
and J.C. DE JON@
‘Laboratory of Virology. Pubiic &a&h Service of the City o~Rotterda~, R~ks~nstitaut (Accepted
voor VoIksgezondhe~d en Mi~ie~hygi~ne, B&haven.
23 August
Fastidious extracted
DNA and
SmaI treatment.
in faecal
rapid
adenoviruses
samples
method.
Fastidious
were too small it was not always
fastidious
Rotierda~.
‘Laboratory o~V~ro~ogy~
The ~ethertands
1984)
adenovirus
by a simple
endonuclease
40 AND 41
adenovirus
possible
40 and 41
obtained
The extracted
DNA was detected
to identify
DNA
from children viral DNA
with acute gastroenteritis
was characterized
was
by restriction
in 58 of 65 cases. If faecal samples
virus DNA.
restriction
extraction
endonuclease
patterns
genome
variants
INTRODUCTION
Human adenoviruses can play a role in viral gastroenteritis in young children as is suggested by their presence in the stools from such patients, In many cases these adenoviruses, in contrast to most human adenoviruses, could not be propagated in conventional cell cultures (Flewett et al., 1975; Richmond et al., 1979). More recently they were successfully propagated in Chang cells (Kidd et al., 1981), in Graham 293 cells (Takiff et al., 1981), in tertiary monkey kidney cells and in certain sublines of HeLa and HEp-2 celfs {De Jong et al., 1983). These adenoviruses were therefore called ‘fastidious’ adenoviruses (Kidd et al., 1981). Controlled examinations revealed that in particular these fastidious adenoviruses could be etiologically related to acute enteric disease (Brandt et al., 1979). The fastidious adenoviruses were grouped into two new serotypes: 40 and 41 (Johansson et al., 1980; De Jong et al., 1983; Uhnoo et al., 1983). Analysis of the DNAs from adenoviruses 40 and 41 (Ad40 and Ad41) by restriction endonuclease digestion showed a large difference between the genomes of the two serotypes (Kidd et al., 1983; Uhnoo et al., 1983). Fastidious adenoviruses can be
Address reprinz requests to: Dr. J. Buitenwerf, Rotterdam,
Schiedamsedijk
0166-0934/85/$03.30
Laboratory
95, 3011 EN Rotterdam,
ofvirology,
Public Health
The Netherlands.
0 1985 Elsevier Science Publishers
B.V. (Biomedical
Division)
Service of the City of
40
present
in enormous
Svensson
amounts
in stools from patients
et al., 1983). This enabled
ruses from stool and extract the adenovirus clease
analysis
serotyping
(Wade11 et al.,
is time consuming,
the adenovirus clease analysis MATERIALS
with diarrhoea
Wade11 and coworkers
DNA for subsequent
1980). Because
we decided
(Flewett,
restriction
the propagation
to develop a rapid method
and
endonu-
subsequent
for isolation
DNA directly from stool and typing the virus by restriction of the DNA. In this paper we present the results.
AND
1976;
to purify such adenovi-
of
endonu-
METHODS
Fuecal specimens Stool specimens were collected from children (aged O-4 yr) with acute gastroenteritis. The presence of adenovirus in the faecal specimens was shown either by electron microscopy or by growth in HeLa cell cultures or human embryonic lung fibroblasts. Only adenoviruses which could not be propagated readily in HeLa cells or in human embryonic lung fibroblasts were used for serotyping and for restriction endonuclease analysis. Serology Serological viously
typing
described
of the adenoviruses
was done by serum
neutralization
as pre-
by De Jong et al. (1983).
Virus purt~ic~tio~ and DNA extraction For the purification of the adenoviruses from faeces we used the method described by Buitenwerf et al. (1983) with some modifications. Briefly, faeces (approximately 500 ~1) were mixed with 1500 pl sodium-Tris-EDTA 0.005 (Tris-HCl 50 mM pH 7.4, NaCl 100 mM and EDTA 5 mM) and vigorously shaken with glass beads. Thereafter the samples were centrifuged for 10 min at 2,000 X g and 20°C. The supernatant was extracted with an equal volume of trichlorotrifluoroethane (Merck, Darmstadt, F.R.G.) and centrifuged for 10 min at 2,000 X g and 20°C. The aqueous phase was collected and the virus particles were precipitated by the addition of polyethylene glycol6,OOO (10% w/v, Merck, Darmstadt, F.R.G.) and NaCl(0.5 M). They were left standing
for at least 2 h at 4’C and were then centrifuged
for 20 min at 20,000 Xg and
4’C. The pellet was resuspended in 500 pl sodium-Tris-EDTA 0.005 and the virions were lysed by the addition of sodium dodecyl sulphate to 1% w/v (23 pl of a 22% w/v solution, Serva, Heidelberg, F.R.G.). After 10 min at room temperature the sample was extracted with an equal volume of a phenol-chloroform mixture (1 : 1, saturated with sodium-Tris-EDTA 0.005) and centrifuged in an Eppendorf centrifuge for 2 min at room temperature. The aqueous phase containing the viral DNA was collected and subsequently Na-acetate (60 ul of 4 M) and 2 volumes absolute ethanol were added to precipitate the DNA. After at least 2 h at -20°C the DNA was sedimented by centrifugation at 20,000 X g for 30 min at
41
-5°C.
The pellet was vacuum
EDTA
0.001 (Tris-HCI
dialyzed
dried and the DNA
10 mM pH 7.4 and EDTA
for 15 min at 20°C through
urn, Millipore
SA, Molsheim,
1980). The DNA samples
France)
were stored
a Millipore floating
was resuspended
in 25 ul Tris-
1 mM). Thereafter
the DNA was
filter (VSWPO1300
pore size 0.025
on aqua bidest (Marusyk
and Sergeant,
at 4°C.
Restriction endonuclease treatment Eight microliters of the DNA samples
were mixed with 1 pl enzyme buffer (Tris-
HCI 60 mM pH 8.0; KC1 20 mM; MgCl, 60 mM and P-mercaptoethanol60 mM) and 1 1.11restriction endonuclease SmaI (12 units/$; Boehringer, Mannheim, F.R.G.). The mixture was incubated for 1.5 hat 25°C and 0.5 hat 37°C. Electrophoresis of the DNA was performed at 50 V for 1.5-2 h at room temperature in an agarose mini-gel (1% w/v agarose, in Tris 40 mM-acetate 20 mM pH 7.6; EDTA 2 mM and ethidium bromide 10 ug/ml) using a Bio-Rad Mini-Sub Cell (Bio-Rad Laboratories, U.S.A.). The gels were photographed under UV-illumination (254 nm UV tubes, Gelman Sciences Inc, Ann Arbor, MI, U.S.A.) on Polaroid type 665 film using a Kodak 23A filter and a PAL filter (h,,,
590 nm, Schott
Glaswerke,
Mainz,
F.R.G.).
RESULTS
Stool specimens containing fastidious adenoviruses detected by electron microscopy and showing poor growth in cell cultures were used for neutralization tests and DNA analyses. Fig. 1 shows that restriction endonuclease SmaIcaneasilydifferentiate Ad40 and Ad41 DNA extracted directly from stool. Furthermore it is obvious that DNA extracted from Ad41 may show two different restriction endonuclease patterns as was earlier observed by Wade11 (personal communication) and Kidd et al. (1983). These patterns
will be called Ad41P (pattern
of prototype
virus Tak) and Ad4la.
In another series of experiments, instead of using the faecal sample we used infected cell cultures as a source of adenovirus DNA. One conventional tube culture proved to yield sufficient DNA for typing the adenovirus (data not shown). Since in some patients
both adenovirus
and rotavirus
particles
are present
in the
faeces we examined the influence of the presence of rotavirus RNA on the patterns found for the adenovirus DNA treated with SmaI. Both the adenovirus DNA fragments
and the rotavirus
RNA segments
could
be distinguished
in the gel (Fig. 2).
Therefore the presence of rotavirus RNA does not interfere with the typing of adenoviruses by the method described here. In Table 1 the serological differentiation method is compared with the restriction enzyme analysis for a number of fastidious adenoviruses from stool specimens. There was a close correlation between serological and biochemical differentiation. However, in 7 of 65 specimens (10.7%) the amount of faeces was insufficient for reliable DNA analysis, no bands being visualized. Furthermore, the method using the restriction enzyme SmaI offers the possibility for detection of genome variants (Ad41 and
Fig. 1. Smal digest of adenovirus
DNA, extracted
during
serotypes
1.5 h at 50 V. Adenovirus
from various
stools, separated
on a 1% (w/v) agarose
gel
shown are: 40 (lane 2): 41P (lanes 4 and 7) and 41a (lanes 1,3,5.
6, 8 and 9). Fig. 2. Electrophoresis adenovirus
of adenovirus
and rotavirus,
DNA and rota RNA treated SmaI (3) and untreated
TABLE
DNA and rotavirus
on a 1% (w/v) agarose
RNA, extracted
gel during
with SmaI (1) and untreated
from
a stool carrying
both
2 h at 50 V. Lanes 1 and 2: mixture of adeno
(2). Lanes 3 and 4: adeno 41a DNA treated
with
(4).
1
Comparison
between
neutralization
serotypes
40 and 41 in stoolsa
Serotype
by
and restriction
endonuclease
Serotype
by restriction
endonuclease
40
4IPb
41a’
analysis
analysis
neutralization
40
16
41
neg* 4
2
22
3
Not typed
6
-
12
-
Total (65)
22
2
34
7
a Stools were selected by electron b Adenovirus ’ Genome
41 prototype
variant
of Ad41.
d No DNA bands visible.
(Tak).
microscopy
on the presence
of adenovirus
(SmaI) of fastidious
adenovirus
43
Ad4la).
In 18 cases in which serological
not done the restriction
endonuclease
differentiation
between
SmaI gave clear-cut
Ad40 and Ad41 was
results.
DISClJSSION
In this paper we present data that show how a simple and rapid method can be used to isolate and type human fastidious adenovirus DNA from faecal samples. The results presented with stool specimens carrying Ad40 or Ad41 showed that in 58 of 65 cases sufficient virus particles were present in the specimens to allow for the isolation of a sufficient amount of DNA for a restriction enzyme analysis. Attempts method for the extraction of adenovirus DNA from stools carrying
to use the same other adenovi-
ruses (e.g. types 2,8,14,17 and 3 I) have failed (data not shown). Probably this was due to the fact that the number of virus particles present in stools containing these species of adenoviruses in general is much less than the number of virus particles present in faecal specimens containing Ad40 or Ad41 (Svensson et al., 1983). The use of the restriction endonuclease SmaI allows for the recognition of genome variants among the adenoviruses: for example Ad 19 and Adl9a (Wade11 and De Jong, 1980) and Ad41 and Adlla (Kidd et al., 1983; this paper). However, in the case of Ad40 and Ad41 these interspecies variations are small and do not interfere with the species determination. When more restriction endonucleases are applied, more genome variants may be detected, as was demonstrated for Ad40 and Ad41 (Kidd, 1984a,b). Our method is very convenient for this kind of research, provided that sufficient virus is present in the stool specimen. In a previous paper (Buitenwerf et al., 1983) we demonstrated the presence of adenovirus DNA in the rotavirus RNA preparations. In Fig. 2 of the present paper we showed that the presence of rotavirus RNA did not hamper the typing of this adenovirus DNA. Although the method adenovirus and rotavirus RNA or DNA),
described is probably less sensitive than the detection of by electron microscopy (empty virus particles will not yield
the results presented
in Fig. 2 and in Table
1 and those presented
in
our previous paper (Buitenwe~et al., 1983) indicate that the method can be used as a diagnostic procedure too. Within approximately IO h the results of the test can be known. REFERENCES
Brandt,
CD..
Chanock, Buitenwerf,
Kim, H.W.,
Yolken,
R.M. and Parrott, J., Muilwijk-van
De Jung. J.C., Wigand, and Firtzloff,
R.G.,
Flewett,
T.M.,
Flewett,
T.M., Bryden,
R.H.,
R.H., Alphen,
Kapikian,
A.Z., Arrobio,
J.O., Rodriguez,
1979, Am. J. Epidemiol.
110, 243.
M. and Schaap,
1983, J. Med. Virol.
R., Kidd, A.H., Wadell, 1983, J. Med. Viral.
C.J.P.,
G., Kapsenbeg,
J.G.,
Muzerie,
11, 215.
1976, Proc. R. Sot. Med. 69. 693. A.S.. Davies,
H. and Morris,
C.A.,
1975, Lancet
1, 4.
W.J., Wyatt,
R.G.,
12, 71.
C.J., Wermenbol,
A.G.
44
Johansson,
M.E., Uhnoo,
Kidd. A.H.,
I., Kldd, A.H., Madeley,
1984a, J. Med. Viral.
14. 49. 14, 235.
Kidd, A.H.,
1984b, J. Med. Viral.
Kidd,
A.H..
Chrystie,
Kidd,
A.H.,
Banatvala,
Marusyk,
I.L.. Banatvala,
J.E. and Hawkins,
J.E. and de Jong,
R. and Sergeant,
Richmond,
C.R. and Wadell, G., 1980, J. Clin. Microbial.
A., 1980, Anal.
J.C.,
G., 1981, Lancet
1983, J. Med. Virol.
Biochem.
S.J., Gaul, E.O., Dunn, SM., Ashley. CR.,
12,95.
2. 371.
1 I, 333.
105, 403. Clarke,
S.K.R. and Seymour.
N.R., 1979, Lancet 1,
1178. Svensson,
L., Wadell, G., Uhnoo,
Takiff,
H.E., Straus,
Uhnoo,
I., Wadell,
Wadell,
G. and de Jong,
Wadell,
G., HammarskjBld,
354, 16.
I.. Johansson,
S.E. and Garon, G., Svensson, J.C.,
C.F.,
M. and Von Bonsdorff, 1981, Lancet
L. and Johansson,
1980, Infect.
M.L., Winberg,
Immun.
C.H., 1983, J. Gen. Virol. 64,25 17.
2, 832.
M., 1983, Dev. Biol. Standard.
55, 311.
27, 292.
G.. Varsany,
T. and Sundell,G., 1980,Ann. N.Y. Acad. Sci.
39
10 (1985) 39-44
Elsevier JVM 00356
A SIMPLE AND RAPID METHOD WITHOUT
JOHANNES
FOR TYPING ADENOVIRUSES
CULTIVATION
BUITENWERF’,
J.J. LOUWERENS’
and J.C. DE JON@
‘Laboratory of Virology. Pubiic &a&h Service of the City o~Rotterda~, R~ks~nstitaut (Accepted
voor VoIksgezondhe~d en Mi~ie~hygi~ne, B&haven.
23 August
Fastidious extracted
DNA and
SmaI treatment.
in faecal
rapid
adenoviruses
samples
method.
Fastidious
were too small it was not always
fastidious
Rotierda~.
‘Laboratory o~V~ro~ogy~
The ~ethertands
1984)
adenovirus
by a simple
endonuclease
40 AND 41
adenovirus
possible
40 and 41
obtained
The extracted
DNA was detected
to identify
DNA
from children viral DNA
with acute gastroenteritis
was characterized
was
by restriction
in 58 of 65 cases. If faecal samples
virus DNA.
restriction
extraction
endonuclease
patterns
genome
variants
INTRODUCTION
Human adenoviruses can play a role in viral gastroenteritis in young children as is suggested by their presence in the stools from such patients, In many cases these adenoviruses, in contrast to most human adenoviruses, could not be propagated in conventional cell cultures (Flewett et al., 1975; Richmond et al., 1979). More recently they were successfully propagated in Chang cells (Kidd et al., 1981), in Graham 293 cells (Takiff et al., 1981), in tertiary monkey kidney cells and in certain sublines of HeLa and HEp-2 celfs {De Jong et al., 1983). These adenoviruses were therefore called ‘fastidious’ adenoviruses (Kidd et al., 1981). Controlled examinations revealed that in particular these fastidious adenoviruses could be etiologically related to acute enteric disease (Brandt et al., 1979). The fastidious adenoviruses were grouped into two new serotypes: 40 and 41 (Johansson et al., 1980; De Jong et al., 1983; Uhnoo et al., 1983). Analysis of the DNAs from adenoviruses 40 and 41 (Ad40 and Ad41) by restriction endonuclease digestion showed a large difference between the genomes of the two serotypes (Kidd et al., 1983; Uhnoo et al., 1983). Fastidious adenoviruses can be
Address reprinz requests to: Dr. J. Buitenwerf, Rotterdam,
Schiedamsedijk
0166-0934/85/$03.30
Laboratory
95, 3011 EN Rotterdam,
ofvirology,
Public Health
The Netherlands.
0 1985 Elsevier Science Publishers
B.V. (Biomedical
Division)
Service of the City of
40
present
in enormous
Svensson
amounts
in stools from patients
et al., 1983). This enabled
ruses from stool and extract the adenovirus clease
analysis
serotyping
(Wade11 et al.,
is time consuming,
the adenovirus clease analysis MATERIALS
with diarrhoea
Wade11 and coworkers
DNA for subsequent
1980). Because
we decided
(Flewett,
restriction
the propagation
to develop a rapid method
and
endonu-
subsequent
for isolation
DNA directly from stool and typing the virus by restriction of the DNA. In this paper we present the results.
AND
1976;
to purify such adenovi-
of
endonu-
METHODS
Fuecal specimens Stool specimens were collected from children (aged O-4 yr) with acute gastroenteritis. The presence of adenovirus in the faecal specimens was shown either by electron microscopy or by growth in HeLa cell cultures or human embryonic lung fibroblasts. Only adenoviruses which could not be propagated readily in HeLa cells or in human embryonic lung fibroblasts were used for serotyping and for restriction endonuclease analysis. Serology Serological viously
typing
described
of the adenoviruses
was done by serum
neutralization
as pre-
by De Jong et al. (1983).
Virus purt~ic~tio~ and DNA extraction For the purification of the adenoviruses from faeces we used the method described by Buitenwerf et al. (1983) with some modifications. Briefly, faeces (approximately 500 ~1) were mixed with 1500 pl sodium-Tris-EDTA 0.005 (Tris-HCl 50 mM pH 7.4, NaCl 100 mM and EDTA 5 mM) and vigorously shaken with glass beads. Thereafter the samples were centrifuged for 10 min at 2,000 X g and 20°C. The supernatant was extracted with an equal volume of trichlorotrifluoroethane (Merck, Darmstadt, F.R.G.) and centrifuged for 10 min at 2,000 X g and 20°C. The aqueous phase was collected and the virus particles were precipitated by the addition of polyethylene glycol6,OOO (10% w/v, Merck, Darmstadt, F.R.G.) and NaCl(0.5 M). They were left standing
for at least 2 h at 4’C and were then centrifuged
for 20 min at 20,000 Xg and
4’C. The pellet was resuspended in 500 pl sodium-Tris-EDTA 0.005 and the virions were lysed by the addition of sodium dodecyl sulphate to 1% w/v (23 pl of a 22% w/v solution, Serva, Heidelberg, F.R.G.). After 10 min at room temperature the sample was extracted with an equal volume of a phenol-chloroform mixture (1 : 1, saturated with sodium-Tris-EDTA 0.005) and centrifuged in an Eppendorf centrifuge for 2 min at room temperature. The aqueous phase containing the viral DNA was collected and subsequently Na-acetate (60 ul of 4 M) and 2 volumes absolute ethanol were added to precipitate the DNA. After at least 2 h at -20°C the DNA was sedimented by centrifugation at 20,000 X g for 30 min at
41
-5°C.
The pellet was vacuum
EDTA
0.001 (Tris-HCI
dialyzed
dried and the DNA
10 mM pH 7.4 and EDTA
for 15 min at 20°C through
urn, Millipore
SA, Molsheim,
1980). The DNA samples
France)
were stored
a Millipore floating
was resuspended
in 25 ul Tris-
1 mM). Thereafter
the DNA was
filter (VSWPO1300
pore size 0.025
on aqua bidest (Marusyk
and Sergeant,
at 4°C.
Restriction endonuclease treatment Eight microliters of the DNA samples
were mixed with 1 pl enzyme buffer (Tris-
HCI 60 mM pH 8.0; KC1 20 mM; MgCl, 60 mM and P-mercaptoethanol60 mM) and 1 1.11restriction endonuclease SmaI (12 units/$; Boehringer, Mannheim, F.R.G.). The mixture was incubated for 1.5 hat 25°C and 0.5 hat 37°C. Electrophoresis of the DNA was performed at 50 V for 1.5-2 h at room temperature in an agarose mini-gel (1% w/v agarose, in Tris 40 mM-acetate 20 mM pH 7.6; EDTA 2 mM and ethidium bromide 10 ug/ml) using a Bio-Rad Mini-Sub Cell (Bio-Rad Laboratories, U.S.A.). The gels were photographed under UV-illumination (254 nm UV tubes, Gelman Sciences Inc, Ann Arbor, MI, U.S.A.) on Polaroid type 665 film using a Kodak 23A filter and a PAL filter (h,,,
590 nm, Schott
Glaswerke,
Mainz,
F.R.G.).
RESULTS
Stool specimens containing fastidious adenoviruses detected by electron microscopy and showing poor growth in cell cultures were used for neutralization tests and DNA analyses. Fig. 1 shows that restriction endonuclease SmaIcaneasilydifferentiate Ad40 and Ad41 DNA extracted directly from stool. Furthermore it is obvious that DNA extracted from Ad41 may show two different restriction endonuclease patterns as was earlier observed by Wade11 (personal communication) and Kidd et al. (1983). These patterns
will be called Ad41P (pattern
of prototype
virus Tak) and Ad4la.
In another series of experiments, instead of using the faecal sample we used infected cell cultures as a source of adenovirus DNA. One conventional tube culture proved to yield sufficient DNA for typing the adenovirus (data not shown). Since in some patients
both adenovirus
and rotavirus
particles
are present
in the
faeces we examined the influence of the presence of rotavirus RNA on the patterns found for the adenovirus DNA treated with SmaI. Both the adenovirus DNA fragments
and the rotavirus
RNA segments
could
be distinguished
in the gel (Fig. 2).
Therefore the presence of rotavirus RNA does not interfere with the typing of adenoviruses by the method described here. In Table 1 the serological differentiation method is compared with the restriction enzyme analysis for a number of fastidious adenoviruses from stool specimens. There was a close correlation between serological and biochemical differentiation. However, in 7 of 65 specimens (10.7%) the amount of faeces was insufficient for reliable DNA analysis, no bands being visualized. Furthermore, the method using the restriction enzyme SmaI offers the possibility for detection of genome variants (Ad41 and
Fig. 1. Smal digest of adenovirus
DNA, extracted
during
serotypes
1.5 h at 50 V. Adenovirus
from various
stools, separated
on a 1% (w/v) agarose
gel
shown are: 40 (lane 2): 41P (lanes 4 and 7) and 41a (lanes 1,3,5.
6, 8 and 9). Fig. 2. Electrophoresis adenovirus
of adenovirus
and rotavirus,
DNA and rota RNA treated SmaI (3) and untreated
TABLE
DNA and rotavirus
on a 1% (w/v) agarose
RNA, extracted
gel during
with SmaI (1) and untreated
from
a stool carrying
both
2 h at 50 V. Lanes 1 and 2: mixture of adeno
(2). Lanes 3 and 4: adeno 41a DNA treated
with
(4).
1
Comparison
between
neutralization
serotypes
40 and 41 in stoolsa
Serotype
by
and restriction
endonuclease
Serotype
by restriction
endonuclease
40
4IPb
41a’
analysis
analysis
neutralization
40
16
41
neg* 4
2
22
3
Not typed
6
-
12
-
Total (65)
22
2
34
7
a Stools were selected by electron b Adenovirus ’ Genome
41 prototype
variant
of Ad41.
d No DNA bands visible.
(Tak).
microscopy
on the presence
of adenovirus
(SmaI) of fastidious
adenovirus
43
Ad4la).
In 18 cases in which serological
not done the restriction
endonuclease
differentiation
between
SmaI gave clear-cut
Ad40 and Ad41 was
results.
DISClJSSION
In this paper we present data that show how a simple and rapid method can be used to isolate and type human fastidious adenovirus DNA from faecal samples. The results presented with stool specimens carrying Ad40 or Ad41 showed that in 58 of 65 cases sufficient virus particles were present in the specimens to allow for the isolation of a sufficient amount of DNA for a restriction enzyme analysis. Attempts method for the extraction of adenovirus DNA from stools carrying
to use the same other adenovi-
ruses (e.g. types 2,8,14,17 and 3 I) have failed (data not shown). Probably this was due to the fact that the number of virus particles present in stools containing these species of adenoviruses in general is much less than the number of virus particles present in faecal specimens containing Ad40 or Ad41 (Svensson et al., 1983). The use of the restriction endonuclease SmaI allows for the recognition of genome variants among the adenoviruses: for example Ad 19 and Adl9a (Wade11 and De Jong, 1980) and Ad41 and Adlla (Kidd et al., 1983; this paper). However, in the case of Ad40 and Ad41 these interspecies variations are small and do not interfere with the species determination. When more restriction endonucleases are applied, more genome variants may be detected, as was demonstrated for Ad40 and Ad41 (Kidd, 1984a,b). Our method is very convenient for this kind of research, provided that sufficient virus is present in the stool specimen. In a previous paper (Buitenwerf et al., 1983) we demonstrated the presence of adenovirus DNA in the rotavirus RNA preparations. In Fig. 2 of the present paper we showed that the presence of rotavirus RNA did not hamper the typing of this adenovirus DNA. Although the method adenovirus and rotavirus RNA or DNA),
described is probably less sensitive than the detection of by electron microscopy (empty virus particles will not yield
the results presented
in Fig. 2 and in Table
1 and those presented
in
our previous paper (Buitenwe~et al., 1983) indicate that the method can be used as a diagnostic procedure too. Within approximately IO h the results of the test can be known. REFERENCES
Brandt,
CD..
Chanock, Buitenwerf,
Kim, H.W.,
Yolken,
R.M. and Parrott, J., Muilwijk-van
De Jung. J.C., Wigand, and Firtzloff,
R.G.,
Flewett,
T.M.,
Flewett,
T.M., Bryden,
R.H.,
R.H., Alphen,
Kapikian,
A.Z., Arrobio,
J.O., Rodriguez,
1979, Am. J. Epidemiol.
110, 243.
M. and Schaap,
1983, J. Med. Virol.
R., Kidd, A.H., Wadell, 1983, J. Med. Viral.
C.J.P.,
G., Kapsenbeg,
J.G.,
Muzerie,
11, 215.
1976, Proc. R. Sot. Med. 69. 693. A.S.. Davies,
H. and Morris,
C.A.,
1975, Lancet
1, 4.
W.J., Wyatt,
R.G.,
12, 71.
C.J., Wermenbol,
A.G.
44
Johansson,
M.E., Uhnoo,
Kidd. A.H.,
I., Kldd, A.H., Madeley,
1984a, J. Med. Viral.
14. 49. 14, 235.
Kidd, A.H.,
1984b, J. Med. Viral.
Kidd,
A.H..
Chrystie,
Kidd,
A.H.,
Banatvala,
Marusyk,
I.L.. Banatvala,
J.E. and Hawkins,
J.E. and de Jong,
R. and Sergeant,
Richmond,
C.R. and Wadell, G., 1980, J. Clin. Microbial.
A., 1980, Anal.
J.C.,
G., 1981, Lancet
1983, J. Med. Virol.
Biochem.
S.J., Gaul, E.O., Dunn, SM., Ashley. CR.,
12,95.
2. 371.
1 I, 333.
105, 403. Clarke,
S.K.R. and Seymour.
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