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Microbiol. Immunol., 39(10), 825-828, 1995

Detection of Astroviruses from Stool Samples in

Japan Using Reverse Transcription and Polymerase
Chain Reaction Amplification

Kunihiro Saito1,2,Hiroshi Ushijima*,1, Osamu Nishio1, Mituaki Oseto3, Hiroshi Motohiro4,

Yuichi Ueda5, Michio Takagi5, Shigekazu Nakaya5, Tamie Ando6, Rodger Glass6,
and Kohji Zaiman2
1
Departmentof Microbiology,Institute of Public Health, Minato-ku,Tokyo108, Japan, 2Department of Clinical Nutrition, Tokyo
UniversityofAgriculture, Setagaya-ku, Tokyo156, Japan, 3Division of Virus,Ehime Prefectural Institute of Public Health, Ma-
tsuyama,Ehime 790,Japan, 4Department of Pediatrics, Kurume UniversitySchool of Medicine,Kurume, Fukuoka 830, Japan,
'Department of Pediatrics , Maizuru-KyosaiHospital, Maizuru, Kyoto 625, Japan, and 6Viral Gastroenteritis Section, Centers
for Disease Control and Prevention, 1600 Clifton Road NE, Atlanta, Georgia 30333, U.S.A.

ReceivedApril 27, 1995. AcceptedJune 20, 1995

Abstract: We developed a reverse transcription and polymerase chain reaction (RT-PCR) method for
detecting astrovirus serotypes 1, 2, 3, 5, 6 and 7 (but not serotype 4). Furthermore, we developed the spe-
cific primers for detecting serotypes 1 and 2, the most predominant serotypes in the world. Sensitivity of
the first PCR with serotype common primers was about 10 times higher than that of enzyme immunoassay
with monoclonal antibody (EIA-MAb). Sensitivity of the second PCR with the serotype-specific primers
was even higher. The RT-PCR method was useful for detecting astroviruses from clinical samples, especially
serotypes 1 and 2.

Key words: Astrovirus, RT-PCR, Enzyme immunoassay, Epidemiology

Epidemiologicstudy of astrovirus has been conduct- prised 53 diarrhealsamples obtainedfrom the outpatient
ed mainly by electron microscopy(EM) (8). Polyclonal pediatric clinic of Maizuru Kyosai Hospital located in
antisera and a group-specific monoclonal antibody centralJapan in the winter of 1992 and 1993;the second
against cultured astrovirushave been developed recent- comprisedsamples collectedat outpatientpediatric clin-
ly for detecting astroviruses by enzyme immunoassay ics and kindlyprovidedby prefecturalinstitutesof health
(EIA). The method is more sensitive than EM and is (four sporadic cases from Aichi Prefectural Institute of
more specificand suitable for screening of astrovirus(4, Health, one case from the Tokyo Metropolitan Institute
10, 14). However, the use of EIA has been restricted of Health and one case from the Ehime PrefecturalInsti-
because the antibodies are difficult to obtain. tute of Health). Details of ages, clinical symptoms and
Recent cloning and sequencing of astroviruses have the total samples examinedby EM and EIA in groups 1
provided molecular approaches for the detection of and 2 are unknown. These samples were previously
astrovirus(5, 10, 16, 17). We have attempted the detec- determinedto be rotavirus-and enteric adenovirus-neg-
tion and serotyping of astrovirus in stool samples in ative by the latex agglutinationtest and EIA, as reported
Japan. The results are comparedwith those obtainedby previously (12, 13).
EIA. Viral RNA was extracted from stool specimenswith
Cultured astroviruses (15), OxAsT1 (serotype 1), guanidine thiocyanateand glass powder as in previous
OxAsT2 (serotype 2), 2684-90 (serotype 3), OxAsT4 reports on molecularepidemiologicalstudy of rotavirus
(serotype4), OxAsT5(serotype5), Symonds(serotype6) infection (12, 13). The RNA was reverse transcripted
and Rana (serotype 7) on Caco 2 cells or LLCMK2 with the desired first polymerase chain reaction (PCR)
cells were used as standard astroviruses. They were primer mixture, deoxyribonucleosidetriphosphatemix-
kindly donated by Dr. Lee (7) and Dr. Glass. Stool ture, avian myeloblastosis virus reverse transcriptase,
specimenswere collected from 2 groups: the first com- Abbreviations: ETA, enzyme immunoassay; EM, electron
*
Address correspondence to Dr. Hiroshi Ushijima, Depart- microscopy; IEM, immune electron microscopy; MAb, mono-
ment of Microbiology, Institute of Public Health, 4-6-1 Shi- clonal antibody; N, negative; P, positive; PCR, polymerase chain
rokanedai, Minato-ku, Tokyo 108, Japan. reaction; RT, reverse transcription.

825
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826 K. SAITO ET AL

reaction buffer and ribonuclease inhibitor at 37 C for 1 hr

(10-13). The first PCR was conducted with 1 U Taq

DNA polymerase (Promega Corporation, Madison,

Wisc., U.S.A.) and the reversed product for 35 cycles (94

C, 1-min denaturing; 45 C, 2-min annealing; 72 C, 3-min

extension; and 72 C, 7-min final extension). The second

PCR was conducted with the desired second PCR primer

mixture, deoxyribonucleoside triphosphate mixture, Taq

DNA polymerase, reaction buffer and the first PCR

product in the same cycles as described above. Both the

first and second PCR products were electrophoresed in

agarose gel containing ethidium bromide and pho-

tographed under UV light.

For the primers used for the first PCR, Beg and End Fig. 1. First PCR analysis of cultured astrovirus serotypes 1 to 7

were chosen from the reported serotype 1 (17) and using primers of Beg and End (see text). From the left side,

serotype 2 (10): Beg 5'-ACC GTG TAA CCC TCC Hinfl-digested ƒÓX 174 DNA size marker (M), serotypes 1 to 7

standard astroviruses, OxAsT1, OxAsT2, 2684-90, OxAsT4,

TCT C-3' (6495-6513 in Astl or 6477-6495 in Ast2);
OxAsT5, Symonds and Rana, respectively. Serotype 4 was not
End 5'-TCC TAC TCG GCG TGG CCG C-3' (6735-
detected by this method.
6717 in Astl or 6717-6699 in Ast2). For the second

PCR for serotype determination, primer mixtures of Si,

S2 and End were chosen: Si 5'-AAC CAA GGA ATG

ACA ATG AC-3' (6524-6543 in Ast1); S2 5'-ACC

TGC GCT GAG AAA CTG-3' (6560-6577 in Ast2).

For EIA, protein A-purified immunoglobulin origi-

nating from hyperimmunization with astrovirus 2 was

coated on the bottom of microplate. After adding stool

suspension and storing overnight, monoclonal antibody

(8E7) was added as a detector. Then an anti-mouse

IgG-horseradish peroxidase conjugate, ortho-phenylene

diamine and 1 N H2SO4 were added in order (3). The

samples were considered positive when P- N was•„0.07

and P/N was •„2, where P is the average absorbance of

2 test wells which were coated with 8E7 and N is the

average absorbance of 2 control wells which were coat- Fig. 2. Second PCR analysis of cultured astrovirus serotypes 1 to

ed with skim milk (9). 7 using primers of Si, S2 and End (see text). From the left side,

serotypes 1 to 7 standard astroviruses, OxAsT1, OxAsT2, 2684-

Virus pellets ultracentrifuged at 100,000 X g for 1 hr
90, OxAsT4, OxAsT5, Symonds, Rana, and Hinfl-digested ƒÓX
through 1 ml of 35% sucrose in phosphate-buffered
174 DNA size marker (M), respectively. Serotype 1 astrovirus was
saline (PBS) were negatively stained with 1% uranyl
specifically amplified by primers of S1 and End. Serotype 2
acetate (pH 4.8) and viewed in an electron microscope.
astrovirus was specifically amplified by primers of S2 and End.
Although the first PCR detected cultured astroviruses

1, 2, 3, 5, 6 and 7, it did not detect serotype 4. The mo-

lecular size of serotypes 1, 2, 3, 5 and 7 was the same were conducted in triplicate. The astroviruses were
(241 bp); however, serotype 6 was smaller than the detected in both samples at 102-folddilution by EIA-
other 4 serotypes (Fig. 1). The second PCR detected MAbs, however, they were detected at 103-folddilution
serotype 1 and serotype 2 at each molecular weight by the firstPCR and at more than 107-foldby the second
position, respectively (212 by and 158 bp), but serotypes PCR (Table 1).
3, 4, 5, 6 and 7 were not detected (Fig. 2). Astroviruses were detected in clinical samples from
A stool sample and a culture sample (OxAsT1), pre- Maizuru, Aichi, Tokyo, Norway and Ehime by EM,
viously determined to be astrovirus 1 by reverse tran- EIA, first PCR and second PCR. The results of PCR
scription (RT)-PCR, were sequentially diluted 10-fold. were the same or more sensitive in comparison with
One set of diluted samples was examined by EIA-MAb the results of EM and EIA (Table 2).
and the other set of diluted samples was examined by RT- The existence of 5 serotypes and recently 7 serotypes
PCR (first and second RT-PCR). These experiments has been reported by immunofluorescence tests and

Table 1. Comparison of sensitivity
Table 2. Detection of astrovirus
rotavirus- and nonadenovirus-gastroenteritis
immune electron microscopy (IEM) (6, 7). However,
serotypingof astrovirusesis not easy since monoclonal
antibodiesspecific for each serotype have not yet been
established.
In this study, we attempted to develope a detection
methodfor astroviruses1 to 7 by group-specificprimers
and serotyping by serotype-specific primer pairs like
rotavirusVP7 and VP4 genes by RT-PCR(1, 2, 10, 13).
Primerpairs of Beg and End designed from serotypes 1
and 2 reacted to serotypes 1, 2, 3, 5, 6 and 7 but not
serotype4. Serotype4 reacts weakly with the common
monoclonal antibody prepared in our EIA compared
with serotypes 1, 2, 3, 5, 6 and 7 (data not shown). The
resultscoincidewith recent analysisof the Beg regionof
serotype4 whichshowed a differentnucleotidesequence
from serotypes 1 and 2 (serotype 4 sequence data were
depositedwith GenBankunderaccessionNo. Z33883by
Willcocks).
Serotype 1 and 2 astroviruses account for more than
half of all astrovirusesdetectedby IEM (7). Our primers
Si and S2 reacted specificallywith serotypes 1 and 2,
respectively.These resultsmay indicatethat our RT-PCR
method is useful for detecting astrovirus from stool
specimensand for partial serotyping.
The sensitivity of the first PCR was almost 10-fold
higher compared with that of EIA-MAb. The second
PCR was more sensitive than EIA-MAb. PCR may be
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NOTES 827
by EIA and RT-PCR for detecting astroviruses
by EM, EIA and RT-PCR from patients with nonbacterial-, non-
in Japan
more useful than EIA-MAb in detecting astrovirus from
rotavirus-negative and adenovirus-negative samples.
We thank Dr. B. Grinde, Institute of Public Health, Norway,
Dr. T.W. Lee, Public Health Laboratory, John Radcliffe Hospital,
United Kingdom, and Dr. H. Hayashi, Tokyo Metropolitan Insti-
tute of Health, for donating astroviruses. We thank Dr. J.E. Her-
rmann, Univ. Massachusetts for donating monoclonal antibody
against astrovirus. This study was supported by Grants-in-Aid
from the Ministry of Education, Science and Culture, Japan.
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