JOURNAL OF CLINICAL MICROBIOLOGY,0095-1137/00/$04.0010

Aug. 2000, p. 2955–2961 Vol. 38, No. 8

Copyright © 2000, American Society for Microbiology. All Rights Reserved.

Application of a Reverse Transcription-PCR for Identificationand Differentiation of Aichi Virus, a New Member of the

Picornavirus Family Associated with Gastroenteritis in HumansT. YAMASHITA,* M. SUGIYAMA, H. TSUZUKI, K. SAKAE, Y. SUZUKI, AND Y. MIYAZAKI

Department of Microbiology, Aichi Prefectural Institute of Public Health, 7-6, Nagare,Tsujimachi, Kita-ku, Nagoya, Aichi 462-8576, Japan

Received 4 February 2000/Returned for modification 21 March 2000/Accepted 7 June 2000

Aichi viruses isolated in Vero cells from seven patients in five gastroenteritis outbreaks in Japan, fiveJapanese returning from Southeast Asian countries, and five local children in Pakistan with gastroenteritiswere examined for differentiation based on their reactivities with a monoclonal antibody to a standard strain(A846/88) and a reverse transcription-PCR (RT-PCR) of three genomic regions. The RNA sequences weredetermined for 519 bases of these 17 isolates at the putative junction between the C terminus of 3C and the Nterminus of 3D. The analyses revealed an approximately 90% homology between these isolates, which were thendivided into two groups: group 1 (genotype A) included six isolates from four outbreaks and one isolate froma traveler and group 2 (genotype B) included one isolate from the other outbreak, four isolates from returningtravelers, and all of the isolates from the Pakistani children. Based on the isolate sequences, a primer pair anda biotin-labeled probe were designed for amplification and detection of 223 bases at the 3C-3D junction of Aichivirus RNA in fecal specimens. The Aichi virus RNA was detected in 54 (55%) of 99 fecal specimens from thepatients in 12 (32%) of 37 outbreaks of gastroenteritis in Japan. Of the 12 outbreaks, 11 were suspected to bedue to genotype A. These results indicated that RT-PCR can be a useful tool to detect Aichi virus in stoolsamples and that a sequence analysis of PCR products can be employed to identify the prevalent strain in eachincident.

Aichi virus was first recognized in 1989 as the cause ofoyster-associated nonbacterial gastroenteritis in humans. Theability to grow in cultured cells along with other biologicalproperties suggested that Aichi virus was a member of theenteroviruses. However, none of the enterovirus antisera neu-tralized Aichi virus. Furthermore, a morphological study ofpurified Aichi virus virions indicated that the surface structureis characteristic of a small round virus (28). Recent geneticanalyses performed on Aichi virus revealed that Aichi virusshould be classified as a new type of the Picornaviridae familyrather than any other genus such as Enterovirus, Rhinovirus,Cardiovirus, Aphthovirus, and Hepatovirus, as well as the echo-virus 22 group (24). Recently, this virus was assigned to a newgenus named Kobuvirus in the family Picornaviridae (14).“Kobu” means bump or knob in Japanese, which is derivedfrom the characteristic morphology of the virus particle.

Aichi virus was isolated in Vero cells from 6 (12.3%) of 47patients in five gastroenteritis outbreaks, 5 (0.7%) of 722 Jap-anese travelers returning from tours to Southeast Asian coun-tries and complaining of gastrointestinal symptoms at the quar-antine station of Nagoya International Airport in Japan, and 5(2.3%) of 222 Pakistani children with gastroenteritis (25, 26).In this study, based on the nucleotide sequences of this virus,we developed a reverse transcription-PCR (RT-PCR) methodfor the detection of Aichi virus and describe the antigenic andgenetic analysis of these isolates in order to determine therelationship between Aichi virus isolates in Japan and those inother countries.

Viral gastroenteritis is a common illness, occurring in bothepidemic and endemic forms. Rotaviruses, adenovirus types 40and 41, Norwalk-like viruses, and astroviruses have been rec-ognized as major etiological agents of human gastroenteritis(2–7). Aichi virus was also determined to be one of the caus-ative agents of human gastroenteritis. In the enzyme-linkedimmunosorbent assay (ELISA), 13 (23%) of 47 stool samplesfrom adult patients in five oyster-associated gastroenteritis out-breaks were found to be positive for Aichi virus. However,seroconversion against Aichi virus was observed in 20 (47%) of43 patients involved in these five outbreaks by a neutralizationtest using paired sera (26). These results suggested that theELISA was not sufficient for diagnosis of the Aichi virus in-fection. RT-PCR for Aichi virus was also applied for detectionof the RNA in stool samples to reveal the distinct prevalenceof this virus in gastroenteritis outbreaks.

MATERIALS AND METHODS

Virus. The Aichi virus strains used in this study consisted of A1156/87 andA1258/87 from an oyster-associated gastroenteritis outbreak in March 1988;A844/88, A846/88 (standard strain), and A848/88 from an outbreak in March1989; and A942/89 from an outbreak in December 1989 in Japan as previouslyreported (26). Strain A1471/96 was isolated from a patient with gastroenteritisfrom an outbreak in Aichi Prefecture in January 1997 and also used in this study.Aichi virus strains T132/90, M166/92, N128/91, N1277/91, and N628/92 wereisolated from Japanese travelers returning from Thailand (T), Malaysia (M), andIndonesia (N) who had complained of gastrointestinal symptoms at the quaran-tine station of Nagoya International Airport between 1990 and 1992. P766/90,P803/90, P832/90, P840/91, and P880/90 were isolated from children with gastro-enteritis in Pakistan between 1990 and 1992 (25). All 66 types of enteroviruses(including echovirus types 22 and 23) were obtained from the National Instituteof Infectious Diseases, Tokyo, Japan. Astrovirus (types 1, 2, 3, 4, 5, 6, and 7) wasobtained from O. Nishio, National Institute of Public Health, Tokyo, Japan.

The standard Aichi virus strain, A846/88, was grown in Vero cells and purifiedby CsCl and sucrose density gradient centrifugation, as described elsewhere (28).The purified strain (50 mg/ml) was diluted from 1022 to 10210 and applied for

* Corresponding author. Mailing address: Department of Microbi-ology, Aichi Prefectural Institute of Public Health, Nagare 7-6, Tsuji-machi, Kita-ku, Nagoya, Aichi 462-8576, Japan. Phone: 81-52-911-3111. Fax: 81-52-913-3641. E-mail: tyamashita@hi-ho.ne.jp.

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sandwich ELISA for detection of Aichi virus antigen as described previously(26), and an RT-PCR was developed in this study.

Stool samples. Adult stool specimens examined in this study came from 268subjects from 37 outbreaks of nonbacterial acute gastroenteritis in Aichi Prefec-ture, Japan, between 1987 and 1998. Of the 37 outbreaks of nonbacterial acutegastroenteritis, 5 were confirmed to be associated with Aichi virus by detectionwith ELISA or seroconversion with a neutralizing test as described elsewhere(26). The sensitivity of the RT-PCR was compared with those of ELISA andseroconversion using the samples from these five outbreaks. Stool samples con-taining Norwalk-like virus were determined by an ELISA administered by K.Numata, Sapporo Medical College (20). Stool samples containing hepatitis Avirus and rotavirus were determined at our laboratory by ELISA as describedelsewhere (19, 30). Stool samples were also collected from 60 healthy childrenattending kindergarten. All stool samples were prepared as 10% homogenates inveal infusion broth with 0.5% bovine serum albumin (fraction V; Sigma Chem-ical, St. Louis, Mo.) and centrifuged at 10,000 3 g for 20 min, and the superna-tants were stored at 230°C until the RT-PCR assay.

ELISA. Aichi virus antibody-secreting hybridomas against the standard strain(A846/88) were prepared as described elsewhere (15, 27). The ELISA used tocompare the reactivities to Aichi virus isolates was performed as follows. Ananti-Aichi virus (A846/88) guinea pig antiserum, diluted 1:10,000 in phosphate-buffered saline (PBS), was used as the capture antibody. After a second coating,100 ml of cell-cultured isolates (103 to 104 50% tissue culture infective doses per25 ml) was added and incubated overnight at 4°C. After a wash, 100 ml ofanti-Aichi virus monoclonal antibodies (MAbs), diluted 1:1 3 104 to 1:512 3 104

in PBS-Tween 20 with 1% bovine serum albumin, was added. After incubationfor 2 h at 37°C, the plates were washed, and 100 ml of peroxidase-labeled rabbitanti-mouse immunoglobulin G (Zymed, South San Francisco, Calif.) in PBS-Tween with 1% (bovine serum albumin was added to each well and incubated for2 h at 37°C. For color development, o-phenylenediamine (Wako Chemical,Osaka, Japan) was used. The MAb titer for each of the isolates was defined as thegreatest dilution giving an optical density reading three times greater than that invirus-free wells and with an optical density value greater than 0.2.

Primers used in RT-PCR. The primer pairs A, B, and C were initially desig-nated for RT-PCR of Aichi virus isolates based on the sequence of the Aichivirus genome (accession no. AB010145). The sequences of the primers wereselected randomly from different regions of the viral genome. The oligonucleo-tide primer sequences were selected as follows: A (1321, 59-TGGTCCCGTCTCATGCACTCCGC; 2028, 59-CCGGCATGGAACTGTGAGCCGT) amplifiesa 708-bp region of VP 0, B (5412, 59-ACCTGCGGATCAACGTCACCTC; 5968,59-AGAGTAGGCAGCTTGAGGTTCC) amplifies a 557-bp region from the Cterminus of 2C to the 3A-3B junction, and C (6261, 59-ACACTCCCACCTCCCGCCAGTA; 6779, 59-GGAAGAGCTGGGTGTCAAGA) amplifies a 519-bpregion between the C terminus of 3C and the N terminus of 3D.

RT-PCR. Aichi virus grown in Vero cells and fecal extracts were centrifuged at10,000 3 g for 20 min, and the supernatant was collected for RT-PCR. Asdescribed by Jiang et al. (13), 0.2 ml of fecal extract was mixed with 0.1 ml of 24%polyethylene glycol 6000 and 1.5 M NaCl solution, stored at 4°C overnight, andcentrifuged at 3,000 3 g for 20 min. The pellet was suspended in 0.1 ml of waterfor RT-PCR. Virus RNA was extracted using the TRIZOL LS reagent (GIBCOBRL, Grand Island, N.Y.) followed by isopropanol precipitation. Each nucleicacid was suspended in RT (Boehringer GmbH, Mannheim, Germany) mixturescontaining oligo(dT) 15 (Promega, Madison, Wis.) and random primer pd (N) 9(Takara, Kyoto, Japan) and incubated for 60 min at 37°C. PCR mixtures, con-taining primer pairs, were added directly into each of the RT mixtures, andamplification was performed in a Thermal Cycler 9600 (Perkin-Elmer Cetus,Norwalk, Conn.) for 40 cycles (each cycle was 95°C for 30 s, 55°C for 30 s, and72°C for 1 min). Analysis of the amplification product was performed by agaroseminigel electrophoresis, and the product was confirmed as a distinct band withethidium bromide staining.

Cycle sequencing. Following RT-PCR, amplified products from 17 Aichi virusisolates and two to three positive fecal samples from outbreaks were purified byphenol-chloroform extraction. Purified RT-PCR products were then precipitatedwith ethanol, and pelleted DNA was suspended in Tris-EDTA buffer and intro-duced into a pGM-T vector (Promega). The DNA sequence was determined byusing a SequiTherm LongRead Cycle Sequencing Kit-LC (Epicentre Technolo-gies Corporation, Madison, Wis.) and a Model 4000 automated DNA sequencer(Li-Cor, Inc., Lincoln, Nebr.). The nucleotide sequence was determined at leasttwice in both directions. For sequence alignments, we examined dendrograms,utilizing UPGMA (unweighted pair group method with averages), in a GeneticsComputer Group sequence analysis package.

Southern blot analysis. Following amplification by RT-PCR and DNA se-quencing of 17 Aichi virus isolates, a biotin-labeled probe (AiPrb2, 59-biotin-ACCTTCGAAGGTCTGTGCGG) was synthesized and purified by Life Technol-ogies Oriental, Inc., Tokyo, Japan. PCR products that migrated on agarose minigelelectrophoresis gels were transferred to a nylon membrane (Nytran; Schleicher &Schuell, Dassel, Germany) and irradiated with 120 mJ of UV light (254 nm) inan auto-cross-linker (Stratagene, La Jolla, Calif.). Blots were hybridized in 0.75M NaCl–20 mM Tris-HCl (pH 8.0)–2.5 mM EDTA–1% sodium dodecyl sulfate–Denhardt’s solution (0.2% bovine serum albumin, 0.2% Ficoll, and 0.2% poly-vinylpyrrolidone)–50 mg of salmon sperm DNA per ml at 55°C with AiPrb2.After 20 h of hybridization, all blots were washed with 23 SSC (13 SSC is 0.15

M NaCl plus 0.015 M sodium citrate)–0.1% sodium dodecyl sulfate at 65°C andstained with streptoavidin- and alkaline phosphatase-labeled biotin and an alka-line phosphatase substrate kit (Vector Laboratories, Inc., Burlingame, Calif.).

Nucleotide sequence accession numbers. The sequences described above havebeen deposited in the DDBJ, EMBL, and GenBank databases under accessionno. AB034649 to AB034663.

RESULTS

Reactivity of MAb for isolates. A selected hybridoma, whichproduced an antibody reactive with a prototype strain (A846/88), was designated clone Ai/8. The immunoglobulin subclasswas determined to be immunoglobulin G1. The reactivity ofthe MAb for 17 isolates was examined by ELISA. MAb Ai/8reacted at titers between 1:32 3 104 and 1:128 3 104 with 7 of17 isolates. However, it reacted only weakly with 10 of 17isolates at a titer of 1:2 3 104 or less. These 10 isolates includedone from an outbreak in Japan; four from travelers returningfrom Thailand, Malaysia, and Indonesia; and all of those fromthe Pakistani children (Table 1).

Reactivity for isolates in RT-PCR. The three pairs of prim-ers (A, B, and C) amplified 708, 557, and 519 bp, respectively.Figure 1 shows the results of RT-PCR-amplified products seenin an agarose gel after 40 cycles, using the Aichi virus standardstrain (A846/88) RNA as the template. Seventeen isolates wereexamined by RT-PCR using these three primer sets. Theprimer sets A and B could not amplify the products of oneisolate from an outbreak; four isolates from travelers returningfrom Thailand, Malaysia, and Indonesia; and all of the isolatesof the Pakistani children with which MAb Ai/8 had reacted atlow titers. The primer set C could amplify products from all 17isolates at the putative junction between the C terminus of 3Cand the N terminus of 3D. The sequence analysis of theseproducts revealed approximately 90% homology among the 17isolates. The dendrogram based on these sequences is depictedin Fig. 2, indicating that Aichi virus isolates could be dividedinto two groups (genotypes A and B). Figure 3 shows thesequence alignment of these isolates in the 3C region based onwhich the two genogroups were defined. These groups werealso identified using reactivities for primer sets A and B and inELISA using MAb Ai/8. Genogroup A stimulated a reaction in

TABLE 1. Reactivities for 16 isolates of Aichi virus with MAb Ai/8and RT-PCR for the standard strain

Virus Reactivity forMAb Ai/8b

Reactivity for RT-PCR using primer set Genotype

A B C

A1156/87 32 1 1 1 AA1258/87 32 1 1 1 AA844/88 64 1 1 1 AA846/88a 128 1 1 1 AA848/88 32 1 1 1 AA1471/96 32 1 1 1 AN128/91 32 1 1 1 AA942/89 ,1 2 2 1 BN1277/91 1 2 2 1 BN628/92 1 2 2 1 BT132/90 1 2 2 1 BM166/92 ,1 2 2 1 BP766/90 2 2 2 1 BP803/90 ,1 2 2 1 BP832/90 1 2 2 1 BP840/91 1 2 2 1 BP880/90 1 2 2 1 B

a Standard strain.b Reciprocal titer, (310,000).

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RT-PCR using primer sets A, B, and C and ELISA using MAbAi/8. On the other hand, genogroup B did not stimulate areaction by primer sets A and B and MAb Ai/8 (Table 1).Based on these sequences, a primer pair, C94b-246k (C94b,59-GACTTCCCCGGAGTCGTCGTCT; 246k, 59-GACATCCGGTTGACGTTGAC), and a biotin-labeled probe (AiPrb2)were designated for the detection of 223 bp at the 3C-3Djunction of Aichi virus RNA. These primers were unable toamplify RT-PCR products from Norwalk-like virus, rotavi-ruses, hepatitis A virus, 66 types of enteroviruses (includingechovirus types 22 and 23), and 7 types of astroviruses. Aichi

virus RNA was detected by RT-PCR using the primers C94b-246k followed by Southern blot analysis in a 1028 dilution ofpurified strain A846/88 (50 mg/ml), while a 1024 dilution of thesample was required to show a positive result by sandwichELISA (Fig. 4). Based on the above results, these primers andprobe AiPrb2, used for amplification and identification of theAichi virus RNA from fecal specimens, were found to be ef-fective.

Detection of Aichi virus RNA in stool samples. A total of 268fecal specimens from 37 outbreaks of gastroenteritis in AichiPrefecture, including 21 outbreaks of oyster-associated gastro-enteritis, were examined by RT-PCR using the primer pairC94b-246K. Aichi virus RNA was detected in 54 (20.5%) of268 samples (12 of 37 outbreaks) by the RT-PCR (Table 2).We found that 54 of 99 patients (55%) were Aichi virus posi-tive in the 12 outbreaks. The positive rates ranged from 14 to82%. On the other hand, 167 stool samples from the other 25outbreaks were negative for Aichi virus RNA. Aichi virus wasnot also detected by RT-PCR in stool samples from 60 healthychildren. Eleven of 12 outbreaks positive for Aichi virus wereassociated with oysters. The other outbreak (outbreak 9) oc-curred on a school excursion and was probably caused bysupper at the students’ hotel. Oysters were not contained in thefood items prepared by the hotel. To determine the genotypeof these positive samples, we sequenced 29 PCR products fromisolates from these 12 outbreaks and compared their sequenceswith those from the 17 isolated Aichi viruses. The sequences ofseven stool samples positive for virus isolation were found tobe identical to those of the isolates. Of the 29 samples, 27samples from 11 outbreaks were classified as genotype A andthe other 2, from outbreak 6, were classified as genotype B. In5 (outbreaks 1, 3, 7, 10, and 11) of these 12 outbreaks, bothstool and paired serum samples were collected from 29 pa-tients. Aichi virus was detected by RT-PCR in 19 (65.6%) of 29patients from the five outbreaks (Table 3). This RT-PCR pos-itive rate (65.6%) was higher than that for ELISA (37.9%) orseroconversion (58.6%). All samples positive for Aichi virus byELISA or seroconversion were also positive by RT-PCR.

DISCUSSION

The sequence analysis of PCR products from 17 isolatesshowed that the isolates could be divided into two groups. Onegroup (genotype A) included five isolates from three outbreaksin Japan and one isolate from a traveler returning from Indo-nesia. The other group (genotype B) consisted of one isolatefrom the other outbreak in Japan; four isolates from travelersfrom Indonesia, Thailand, and Malaysia; and all of the isolatesfrom Pakistani children. The ELISA with MAb Ai/8 revealedhigh reactivity only with isolates of genotype A, and this reac-tivity indicated the presence of a group antigen. RT-PCR usingprimer sets A and B also revealed nonreactivity with geno-group B viruses. These results suggested that the sequence inthe P1 and P2 region of the Aichi virus was more diverse thanthat of the 3C-3D region analyzed in this study but that the twogenogroups were still significantly similar in this region. How-ever, there is no evidence that these genotypic differencesaffect other diagnostic procedures such as neutralization test orELISA for detection of Aichi virus antigen (25, 26, 28).

We proposed designating a genotype A and a genotype B ofAichi virus. Following sequencing of 29 PCR products fromstool samples in 12 outbreaks, 27 products from 11 outbreakswere classified as genotype A. This information was valuablefor confirming the prevalent strain in Japan, where genotype Awas suspected to be more prevalent than genotype B by se-quence analysis of 17 isolates. In this study, we were able to

FIG. 1. Ethidium bromide-stained agarose gel of Aichi virus (A846/88 strain)RT-PCR products. Lane 1, 100-bp DNA ladder from GIBCO BRL; lanes 2 to 4,PCR with Aichi virus primer sets A (lane 2), B (lane 3), and C (lane 4). Thenumbers at right indicate the sizes of the RT-PCR products in base pairs.

FIG. 2. Dendrogram of predicted genetic relationships among 17 isolates ofAichi virus by comparison of 519 bases at the putative junction between the Cterminus of 3C and the N terminus of 3D.

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FIG. 3. Sequence of Aichi virus (A846/88) amplified with primer set C and partial alignment of nucleic acid sequences of isolates in the C terminus of the 3C region.Two genotypic groups (A and B) were defined by the boxed sequences. The position of the cleavage site between the 3C and 3D regions is indicated. The primer pairsC and C94b-245K are underlined, and a biotin-labeled probe (AiPrb2) is double underlined.

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identify another group (genotype B) which consisted of oneisolate from an outbreak in Japan; four of five isolates oftravelers from Indonesia, Thailand, and Malaysia; and all ofthe isolates from the Pakistani children. By accumulating moresequencing data for Aichi virus RNA, it may be possible todetermine even more genotypes. In addition, the prevalence ofa certain genotype may be discovered to be related to a specificgeographic region.

Outbreaks of gastroenteritis have been associated with theconsumption of raw oysters (9, 10, 16, 17, 21). In Japan, infec-tion by a small round virus (Norwalk-like virus) has been re-ported in several oyster-associated gastroenteritis outbreaks(11, 12, 22, 23). Using RT-PCR, we were able to detect Aichivirus in 12 (32%) of 37 outbreaks in Japan occurring between1987 and 1998. Aichi virus was not frequently associated withoutbreaks between 1991 and 1998, and its clinical importancewas not clearly defined in this study. However, the positiverates were greater than 50% in 10 of 12 outbreaks (Table 2).This result suggested the possibility of Aichi virus being anetiological agent of these 10 outbreaks. The observation that11 of the 12 PCR-positive specimens came from oyster-asso-

FIG. 4. Detection of Aichi virus by RT-PCR with primers C94b-264k (A) andidentification by Southern blot hybridization with probe AiPrb2 (B). Serial di-lutions (from 1026 to 10210) of Aichi virus (50 mg/ml) were analyzed by agarosegel electrophoresis and stained with ethidium bromide. M, markers; N, negativecontrol.

TABLE 2. Outbreaks of gastroenteritis tested for Aichi virus by RT-PCR using primer pair C94b-264k

Outbreak RT-PCR result

Genotype (n)a

No. Yr Description No. positive/no. tested %

1 1987 Oysters 5/9 55.6 A (3)2 1988 Oysters 5/7 71.4 A (3)3 Oysters 9/11 81.8 A (3)4 1989 Oysters 0/3 05 Banquet 0/4 06 Banquet 0/15 07 Oysters 4/5 80.0 A (2)8 School lunch 0/9 09 School excursion 9/14 64.3 A (3)10 1990 Oysters 2/4 50.0 B (2)11 Oysters 6/11 54.5 A (2)12 School lunch 0/5 013 Oysters 0/5 014 1991 Oysters 4/6 66.7 A (2)15 1992 Oysters 0/7 016 School lunch 0/8 017 Banquet 0/7 018 1993 School lunch 0/10 019 1994 Oysters 2/14 14.3 A (2)20 Delivered lunch 0/4 021 Oysters 0/6 022 School lunch 0/6 023 Delivered lunch 0/7 024 1995 School lunch 0/5 025 1996 Oysters 0/6 026 1997 Oysters 5/8 62.5 A (3)27 Banquet 0/5 028 Banquet 0/3 029 Dormitory supper 0/5 030 Oysters 0/5 031 Oysters 0/7 032 1998 School lunch 0/16 033 Oysters 2/4 50.0 A (2)34 Oysters 2/6 33.3 A (2)35 Oysters 0/4 036 Oysters 0/5 037 Oysters 0/12 0

Total 55/268 20.5 A (27), B (2)

a Genotypes were determined for the limited number of fecal samples.

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ciated outbreaks suggested a correlation between Aichi virusand seafood pollution. During feeding, shellfish such as oysterscan accumulate pathogenic human microorganisms present insewage-polluted seawater (8). Since the entire shellfish is usu-ally consumed along with the gastrointestinal tract, shellfishmay act as passive carriers of microorganisms such as entericbacteria and viruses. The effective control of enteric bacterialdisease spread by shellfish has resulted in the establishment ofbacteriological standards using a coliform and fecal coliformindex as the basis for a certification program. Improper docu-mentation and an insufficient number of studies related to thetransmission of viral disease by shellfish have so far impededprogress in implementing preventive measures (29). In addi-tion, there has been a lack of sensitive techniques to ade-quately research this problem. RT-PCR can be expected to bea useful tool for detection of Aichi virus in shellfish such asoysters. Furthermore, using the primer set C and pair C94b-246K designated in this study, it will be possible to construct anested PCR for the detection of Aichi virus RNA from envi-ronmental samples such as food and water. Epidemiologicalstudies focusing on the presence of Aichi virus in environmen-tal samples will aid in revealing transmission routes.

Aichi virus RNA was also amplified from nine patients inoutbreak 9, which was not associated with oysters (Table 2).This means that Aichi virus can be transmitted by substances

other than oysters. The prevalence rate for Aichi virus anti-body was found to be 7.2% for persons aged 7 months to 4years. The prevalence rate for antibody to Aichi virus increasedwith age, to about 80% in persons 35 years old (26). Theseresults indicated that the Aichi virus is circulating in Japan.However, subclinical infections may be more common thanclinically manifest diseases such as those caused by picornavi-ruses. Many picornaviruses cause several discrete clinical syn-dromes such as paralysis, aseptic meningitis, respiratory andintestinal illnesses, and hepatitis (1, 18, 31). The same picor-navirus may cause more than one syndrome. In our previousstudy, using ELISA, Aichi virus antigen was detected for onlyone patient, who was diagnosed with lower respiratory tractillness (26). In this study, RT-PCR was 10,000 times moresensitive than ELISA for detection of purified Aichi virus. Thedevelopment of RT-PCR for Aichi virus should be of interestfor the clinical study of Aichi virus. RT-PCR may be useful forestablishing a clinical diagnosis of illnesses including gastro-enteritis.

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TABLE 3. Comparison of sensitivities among RT-PCR, ELISA,and seroconversion in five outbreaks

Outbreak no. Patient no.Identification of Aichi virus infection by:

RT-PCR ELISAa Seroconversiona

1 1 2 2 22 2 2 23 2 2 24 2 2 25 1 2 16 1 1 17 1 1 1

3 8 2 2 29 1 1 110 1 1 111 1 2 112 1 1 113 2 2 214 1 1 115 1 1 1

7 16 1 1 117 2 2 218 1 1 119 1 2 120 1 1 1

10 21 1 1 122 2 2 223 2 2 224 1 2 2

11 25 1 2 126 1 2 127 2 2 228 1 2 229 1 2 1

No. positive (%) 19 (65.6) 11 (37.9) 17 (58.6)

a Reference 26.

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