JOURNAL OF CLINICAL MICROBIOLOGY, Oct. 1988, p. 2001-20050095-1137/88/102001-05$02.00/0Copyright © 1988, American Society for Microbiology

Vol. 26, No. 10

Detection of Human Calicivirus Antigen and Antibody byEnzyme-Linked Immunosorbent AssaysSHUJI NAKATA,"2 MARY K. ESTESl.3* AND SHUNZO CHIBA2

Departments of Virology and Epidemiologyl* and Experimental Medicine,' Baylor College of Medicine, Houston,Texas 77030, and Department of Pediatrics, Sapporo Medical College, Sapporo 060, Japan2

Received 15 January 1988/Accepted 14 June 1988

Enzyme-linked immunosorbent assays (ELISAs) were developed to detect human calicivirus (HCV) antigenand antibody to HCV. The ELISAs were specific for HCV and as sensitive as a previously developedradioimmunoassay. These ELISAs were used to search for evidence of HCV infection in the United States,where HCV gastroenteritis has rarely been reported. One hundred sixty-three stool samples collected fromchildren hospitalized with diarrhea were examined; one sample was positive in the ELISA. Typical calicivirusparticles were found in this stool sample, and these particles reacted with a hyperimmune guinea pig anti-HCVserum by immune electron microscopy. The age-related acquisition of antibody to HCV in hospitalized infantsand children (from birth to 19 years old) without gastroenteritis and in healthy adults was also evaluated. Thepattern of acquisition of antibody to HCV was similar to that for group A rotaviruses, namely, beginning ininfancy and becoming 100% by the age of 4 years. These data suggest that HCV is associated with infantilegastroenteritis in the United States, that infections with HCV are common, and that many infections with HCV(Sapporo strain) may not require hospitalization.

Human calicivirus (HCV) is a virus of 28 to 35 nm indiameter with a characteristic morphology (a Star-of-Davidconfiguration), a single major structural protein of apparentmolecular weight 62,000 (22), and an RNA genome (6).Virological and epidemiological investigations carried outduring the past decade, mainly in Japan and in England (3, 4,7, 9-11, 21), have established that HCV is one cause ofgastroenteritis in children and sometimes in adults. Thedevelopment of a radioimmunoassay (RIA) for detection ofHCV antigen and a blocking RIA for detection of antibody toHCV have facilitated further immunological and seroepide-miological studies of HCV infection (14-16). Although theRIA has proven to be highly sensitive, relatively simple, andsuitable for the screening of a large number of samples, itsuse of radioisotopes and requirement for special equipmentlimit its widespread applicability. These limitationsprompted us to convert the RIA to an enzyme-linked im-munosorbent assay (ELISA) capable of detecting HCVantigen and antibody to HCV. Following comparison ofthese test formats, the ELISAs were used in a preliminarysurvey to determine whether HCV infection is associatedwith cases of gastroenteritis in children requiring hospital-ization in Houston, Tex., and to compare the patterns ofacquisition of antibody to HCV and to group A rotavirus.The prevalence of antibody in serum samples from theUnited States, Canada, and the People's Republic of Chinawas also examined.

MATERIALS AND METHODSClinical specimens. One hundred sixty-nine stool speci-

mens were tested by ELISA for HCV. Three of these stoolsamples were obtained in Sapporo, Japan, and were shownto contain HCV by electron microscopy (EM) and RIA; theywere taken from three patients with HCV gastroenteritis (16)and served as positive controls. The other 166 stool samples,obtained from patients with gastroenteritis in the TexasChildren's Hospital, Houston, or elsewhere in the United

* Corresponding author.

States as previously reported (12, 17), were used as negativecontrols or as test samples in the ELISA for HCV. Thesespecimens had been examined for virus by EM or byconventional ELISA for group A rotaviruses or by bothmethods. They included samples positive for group A rota-viruses by EM, conventional ELISA, or genome analysis;samples positive for adenovirus or small round viruses byEM; and samples lacking any detectable virus. Two fecalsamples containing Norwalk virus were obtained from ex-perimentally infected volunteers in Houston, and a third wasa pool of Norwalk agent treated with Formalin, provided byW. D. Cubitt, Public Health Laboratory Service, CentralMiddlesex Hospital, London, United Kingdom. All stoolsamples, except the formalinized Norwalk agent, were sus-pended in 10 mM phosphate-buffered saline (PBS; pH 7.4) toproduce a 10% (wt/vol) suspension. The suspensions wereclarified by centrifugation at 5,000 x g for 10 min, and thesupernatant was extracted with an equal volume of Genetron(trichlorotrifluoroethane; Curtin Matheson, Houston, Tex.).The aqueous phase was stored at 4°C until tested.

Viruses. Hepatitis A virus, poliovirus, echovirus 22, andcoxsackievirus B6 were obtained from T. G. Metcalf, BaylorCollege of Medicine, Houston, Tex. Canine calicivirus wasprovided by W. D. Cubitt. HCV was purified from the threepositive control stool samples described above by CsCIdensity gradient centrifugation (15, 16). Viruses from visiblebands in the CsCI gradients were dialyzed and used as agentsto immunize mice and rabbits and as antigens in the ELISAs.Preimmune and hyperimmune antisera. Three preimmune

guinea pig serum samples were obtained from guinea pigsfrom Elm Hill Breeding Labs, Chelmsford, Mass. Fivepreimmune rabbit serum samples were collected from spe-cific-pathogen-free rabbits from a colony established at theHouston Veterans Administration Medical Center (5).Guinea pig hyperimmune antiserum to HCV was prepared asdescribed previously (15). Hyperimmune mouse antiserumto HCV was prepared by inoculating BALB/c mice withpurified HCV. One intraperitoneal injection was made withantigen in an equal volume of Freund complete adjuvant;

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Freund incomplete adjuvant was used for booster injections.Rabbit hyperimmune serum was prepared by intramuscularinjection of purified HCV mixed with Freund complete orincomplete adjuvant as described above. Guinea pig hyper-immune sera to SA11 (group A rotavirus), adult diarrhearotavirus (group B rotavirus), and porcine pararotavirus(group C rotavirus; Cowden strain) were prepared as de-scribed previously (17).Serum samples. One hundred forty-two serum samples and

five immunoglobulin pools were tested by the blockingELISA (ELISA-BL) for antibody to HCV (Sapporo strain),including the following. (i) Four paired serum samples frompatients with HCV gastroenteritis were obtained during anoutbreak in Sapporo (16). (ii) Fifty-nine serum samplesremaining after routine diagnostic tests had been collectedfrom children (1 month to 19 years of age) without gastroen-teritis who were admitted in 1986 to Texas Children'sHospital. (iii) Ten serum samples were obtained fromhealthy adults in Houston. (iv) Twenty serum samples wereobtained from patients with chronic idiopathic inflammatorybowel disease (Crohn's disease or ulcerative colitis) inHouston and New York, N.Y. (18). (v) Nineteen serumsamples were collected from staff members in the Depart-ment of Virology and Epidemiology, Baylor College ofMedicine, in 1985 and 1986. (vi) Thirty acute-phase serumsamples were obtained from adults with gastroenteritis ofunknown origin in the United States and Canada (from thecollection of the Viral Gastroenteritis Laboratory of theCenters for Disease Control, Atlanta, Ga.) and kindly pro-vided by G. W. Gary. (vii) Five immunoglobulin pools eachfrom 2,000 adult donors from China were kindly supplied byJ.-Z. Xiang, Shanghai Institute of Biological Products, Min-istry of Public Health, People's Republic of China.ELISA for HCV antigen. ELISAs were performed with

96-well polyvinyl chloride flat-bottom microtiter plates (Dy-natech Laboratories, Inc., Alexandria, Va.) as previouslydescribed (17). PBS containing 10% fetal calf serum and 1%bovine serum albumin (BSA) was used as the diluent forstool samples, culture fluids, blocking serum, and detectorantibody. The peroxidase-conjugated goat antibody tomouse immunoglobulin G was diluted in PBS containing 5%normal guinea pig serum and 1% BSA. For the ELISA,duplicate wells of the microtiter plates were each coatedwith 100 pul of a 1:10,000 dilution of either hyperimmuneguinea pig serum to HCV or preimmune guinea pig serumdiluted in 10 mM PBS (pH 7.4), and the plates wereincubated at 4°C overnight. The plates were then washedtwice with PBS containing 0.1% Tween 20 (PBS-T) andblocked with 1% BSA in PBS for 60 min at 37°C. Theresidual blocking fluid was then removed, 50 ,lI of each testsample (2 to 5% final suspension) was added, and the plateswere incubated at 37°C for 2 h or at 4°C overnight. After fivewashings in PBS-T, 50 pli of a 1:10,000 dilution of hyperim-mune mouse antiserum to HCV was added, and the plateswere incubated for 90 min at 37°C. After five washings inPBS-T, 50 pul of a 1:10,000 dilution of peroxidase-conjugatedgoat immunoglobulin G antibody to mouse immunoglobulinG (Hyclone Laboratories, Logan, Utah) was added. Theplates were incubated for 60 min at 37°C and washed fivetimes in PBS-T. A 100-pul portion of ABTS [2,2'-azino-bis(3-ethylbenzthiazoline-6-sulfonate); Sigma Chemical Co., St.Louis, Mo.], in phosphate citrate buffer (pH 4.0) containing1.5 pul of 30% H202 per ml was added to each well, and theplates were incubated for 30 min at room temperature. TheA414 was measured with a micro-ELISA reader (TitertekMultiskan; Flow Laboratories, Inc., McLean, Va.).

For each sample, the results were expressed as the ratio ofthe A414 in wells coated with hyperimmune serum (positive)to the A414 in wells coated with preimmune serum (negative)(P/N ratio). The cutoff value of this system was obtained bytesting 23 stool suspensions lacking virus. The mean P/Nratio for these negative samples was 1.62, with a standarddeviation of 0.37, and the cutoff value was defined as themean plus three standard deviations (i.e., a P/N ratio of>2.7). A P/N ratio of >2.7 and an A414 of >0.1 wereconsidered to indicate a positive reaction. All tests wereperformed in duplicate, and the results were averaged. Theoptimal dilutions of reagents were determined by checker-board titrations with CsCl-purified HCV and 2% stool sus-pensions containing group A rotavirus.ELISA-BL. The presence of HCV antigen detected in a

specimen by ELISA was confirmed by the following block-ing test. Duplicate 25-pul samples of stool suspensions wereadded to wells that had been precoated with either preim-mune or hyperimmune guinea pig serum, and the plates wereincubated at 4°C overnight. After five washings in PBS-T,each microtiter well, coated with preimmune or hyperim-mune serum, was inoculated with 50 pil of an appropriatedilution of acute-phase or convalescent-phase serum from apatient with HCV gastroenteritis and incubated for 2 h at37°C or overnight at 4°C. After two washings, the wellsreceived mouse hyperimmune serum to HCV and weretreated in the same way as described above. A >50%inhibition of the A414 produced by the convalescent-phaseserum compared with the acute-phase serum was consideredto be positive for HCV antigen.

Antibodies to HCV were measured by ELISA-BL essen-tially as described above. Briefly, 25 pul of a standardpreparation of purified HCV antigen or diluent was added tomicrotiter wells precoated with guinea pig hyperimmuneserum to HCV (1:10,000 dilution), and the plates wereincubated at 4°C overnight. After five washings in PBS-T,duplicate 50-pul portions of twofold dilutions (in 10% fetalcalf serum-1% BSA) of the serum to be tested for antibody,beginning with a 1:50 dilution, were added to the microtiterwells, and the plates were incubated at 37°C for 2 h or at 4°Covernight. The serum samples were initially diluted to 1:50 inPÉS containing 10% fetal calf serum and 1% BSA. After twowashings, the wells received mouse hyperimmune serum toHCV (1:10,000 dilution) and were treated as describedabove. In each microtiter plate, an optimal dilution ofconvalescent-phase serum from a patient with HCV ga-stroenteritis, which produced about 90% reduction in theA414, served as a positive control; diluent alone and an

acute-phase serum sample served as negative controls. A>50% inhibition of the A414 produced by the serum samplescompared with the buffer control was considered to bepositive for antibody to HCV. All tests were performed induplicate, and the results were averaged.

RESULTS

Specificity and sensitivity of ELISA to detect HCV. Thespecificity of the ELISA for detection of HCV was initiallyevaluated with 75 stool specimens and five culture fluids(Table 1). All three stool samples containing HCV by EMand RIA were positive for HCV in the ELISA. None of thestool specimens collected from patients infected with rota-virus (25 specimens), enteric adenovirus (21 specimens),Norwalk virus (3 specimens), or no virus (23 specimens) waspositive. The data from testing these specimens lacking viruswere also used to establish the criteria for positive test

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TABLE 1. Specificity of ELISA for distinguishing HCVfrom other viruses

No. of results of ELISASpecimen tested No. of for HCVand viral content specimens False-

Positive Negative positive'

Stool specimensHCV 3 3 0 0Rotavirus 25 0 25 0Adenovirus 21 0 20 1Norwalk virus 3 0 3 0None identified 23 0 23 0

Culture supernatantsCanine calicivirus 1 0 1 0Poliovirus 1 0 1 0Echovirus 22 1 0 i 0Coxsackievirus B6 1 0 1 0Hepatitis A virus 1 0 1 0

a Positive by ELISA but not blocked by human anti-HCV serum.b Includes specimens used to determine the range of negative results.

results. One adenovirus specimen gave a false-positive re-

action in this test, that is, the P/N ratio for this specimenexceeded 2.7 (the value determined to be three standarddeviations above the mean for negative samples tested withboth preimmune and hyperimmune serum). However, thisreactivity was not blocked by the confirmatory blocking test(i.e., by the addition of convalescent-phase serum frompatients with known HCV gastroenteritis). Five culturefluids containing canine calicivirus, poliovirus, hepatitis Avirus, echovirus 22, or coxsackievirus B6 were negative inthe ELISA. Testing of these specimens by the confirmatoryblocking assay did not affect their absorbance readings at all.

1.300-

1.200-1.100-

1.000-0. 5w0o.soo\i0.800- \ \\

0.700-

0.600-

E 0.500_

O 0.400-

0.300'

0.200-

0.100-L

2 5 10 20 40 80 160 320 640 1.280

Reciprocal of Antigen Dilution

FIG. 1. Titration of HCV antigen in stool specimens from threepatients (-) with gastroenteritis caused by HCV and from twopatients (O) with gastroenteritis due to group A rotavirus. A P/N >2.7 and A414 (O.D.) > 0.1 were considered a positive reaction. Theantigen titers of positive samples were 1:640, 1:320, and 1:40.

The sensitivity of the ELISA and RIA for detection ofHCV was compared by testing CsCl-purified HCV seriallydiluted in 10% fetal calf serum-1% BSA (data not shown).Similar sensitivities were obtained in both of the immunoas-says, and endpoint dilutions were 1:25,600 in the RIA and1:12,800 in the ELISA. These dilutions contained approxi-mately 104 and 2 x 104 particles of HCV per well, respec-tively. Figure 1 shows the ELISA results of titrations ofthree HCV-positive stool specimens and two stool speci-mens containing group A rotavirus.Use of the HCV ELISA to detect HCV in stools of hospital-

ized patients with gastroenteritis. The HCV ELISA was nextused in a preliminary survey of stool specimens obtainedfrom hospitalized children for examination for virus particlesby direct EM. A total of 90 stool samples (including 23samples used to determine the range of negative results) thatwere collected from hospitalized infants with diarrhea andthat lacked any detectable virus when examined by routineEM and by ELISA for group A rotavirus were tested by theELISA for HCV antigen. One false-positive and one positivesample were found in this group.

Diagnostic tests detected no bacterial (except enterotoxi-genic or enteropathogenic Escherichia coli), viral, or para-sitic causes for the diarrhea in the child with the HCV-positive stool. The HCV detected by the ELISA in the stoolspecimen from this child was not detected originally bydirect EM examination of the same specimens. The resultsindicate that our ELISA is more sensitive for detection ofvirus than EM, a finding in agreement with earlier studieswith RIA (15). Reexamination of our ELISA-positive sam-ples by immune EM confirmed the presence of typicalcalicivirus particles (Fig. 2).An additional 27 stool samples obtained from hospitalized

children and containing small round viruses when initiallyexamined by EM were also tested by the HCV ELISA.None of these samples were positive; one sample gave afalse-positive reaction.

FIG. 2. Typical positive immune EM results obtained by testinghuman calicivirus isolated in Houston with a guinea pig antiserum toHCV. A Star-of-David configuration is apparent. This sample wasstained with phosphotungstic acid. Bar, 50 nm.

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100

° 60-

'ri

40-

20-

0

50 100 200 400 800 1.600 3.2006.400 1. 25. 51. 102. 204,800 600 200 400 000

Reciprocal of serum dilution

FIG. 3. Titration of serum antibody to HCV in paired serum

samples obtained from patients with gastroenteritis due to HCV.Symbols: O, patient 1, pre-outbreak; *, patient 1, convalescent; A,

patient 2, pre-outbreak; A, patient 2, convalescent; E, patient 3,pre-outbreak; *, patient 3, convalescent; O, patient 4, pre-out-break; *, patient 4, convalescent. A >50% inhibition of opticaldensity produced by the serum samples compared with buffercontrol was considered to be positive for antibody to HCV.

Specificity and sensitivity of ELISA-BL to detect antibody toHCV. The specificity and sensitivity of the ELISA-BL fordetecting antibody to HCV were evaluated by using fourpaired sera, five hyperimmune sera, and eight preimmunesera. Figure 3 shows the titration of serum antibody to HCVin paired serum samples obtained from patients with HCVgastroenteritis. The antibody titers were higher by theELISA than by the RIA (Table 2). Guinea pig and rabbithyperimmune sera to HCV were positive with antibodytiters of 1:320,000 and 1:20,000, respectively. Three guineapig hyperimmune sera to three groups of rotaviruses andeight guinea pig and rabbit preimmune sera were negative(<1:50).

Prevalence of antibody to HCV. A total of 128 serumsamples or immunoglobulin pools were tested by ELISA-BL. The age-related prevalence of antibody to HCV (Sap-poro) in Houston is shown in Table 3. All infants below theage of 3 months had antibody to HCV. On the other hand,only 3 of 12 infants (25%) in the age group 4 to 11 monthspossessed antibodies to HCV. The prevalence of antibodysteadily increased thereafter and reached a maximum inpreschool children (4 to 6 years). The pattern of acquisition

TABLE 2. Comparison of HCV serum antibody titers byELISA and RIA for paired serum samples collected from patients

with gastroenteritis

ELISA titer RIA titerPatient Pre- Convalescent Rise Pre- Convalescent Rise

outbreaka phase (fold) outbreak phase (fold)

1 <50 1,600 >32 <50 800 >162 <50 3,200 >64 <50 800 >163 1,600 51,200 32 400 6,400 164 400 102,400 256 100 25,600 256

a Pre-HCV outbreak serum samples collected from patients with adeno-virus gastroenteritis (2).

TABLE 3. Age-related prevalence of antibody to HCV and groupA rotavirus in serum samples from adults or children without

acute gastroenteritis in Houston

No. No. (%) positive for antibody to:Agetested HCV Group A rotavirus

0-3 mo 14 14 (100) 14 (100)4-11 mo 12 3 (25) 5 (42)1-3 yr 10 6 (60) 9 (90)4-6 yr 5 5 (100) 5 (100)7-11 yr 7 7 (100) 7 (100)12-19 yr il 10 (91) 10 (91)

-20 yr 292 26 (90) 29 (100)

Total 88 71 (81) 79 (90)

a Of these, 10 were from healthy adults in Houston and 19 were from staffmembers of the Department of Virology and Epidemiology, Baylor College ofMedicine.

of antibodies to HCV was similar to that of antibodies togroup A rotaviruses. However, for group A rotaviruses thehighest prevalence of antibody was found in an earlier agegroup than for HCV. A high prevalence of antibody to HCValso was found in serum samples from adults in Canada(80%) and the United States (Arkansas, Hawaii, Texas, andNew York [>90%]) and in immunoglobulin pools from adultsin the People's Republic of China (100%).

DISCUSSION

ELISAs specific for HCV antigen and antibodies to HCVwere developed. These assays were equivalent in sensitivityto the RIAs previously developed (14, 16) as determined bymeasuring titers of HCV antigen and antibody to HCV.When purified HCV antigen was used, the sensitivity of theELISA was estimated to be 4 x 105 particles per ml. Theresults of testing 75 stool specimens and 5 culture superna-tants with the HCV ELISA illustrate the specificity of theassay. One of the four specimens which initially reacted aspotential positives in the antigen ELISA was confirmed tocontain HCV by the ELISA-BL. The false-positive results(<2% of total samples tested) observed with the three othersamples are not unexpected in tests with stool specimens (1).Some false-positive results can be expected because thehyperimmune guinea pig serum was prepared from HCVpurified from stool, and antibodies to some stool compo-nents must be present in this serum. Importantly, no speci-mens remained falsely positive after being retested with theconfirmatory ELISA-BL. In addition, the specimen positiveafter the confirmatory ELISA-BL was shown to containvirus by immune EM.

In the survey of stool samples collected from hospitalizedinfants with gastroenteritis of unknown origin, we found onepositive stool sample that subsequently was shown to con-tain typical calicivirus particles by immune EM with hyper-immune serum to HCV (Sapporo strain). This result indi-cates that caliciviruses with antigens common to HCV(Sapporo) are present in the United States and that they maybe one of the causative agents of gastroenteritis in infants inthe United States. Although the proportion ofHCV detectedin stool samples from hospitalized children in this study waslow (<1%), in agreement with previous reports (15, 19), thehigh prevalence of antibody acquisition suggests that infec-tions with HCV (Sapporo) are common. Since this antibodytest reportedly only detects antibody to HCV (Sapporo) (7),these results suggest that most infections with HCV (Sap-

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HUMAN CALICIVIRUS ANTIGEN AND ANTIBODY ELISAS

poro) may not require hospitalization. Some HCV infectionsrequiring hospitalization also may have been missed, be-cause the antiserum used in this ELISA does not detect allstrains of HCV (7). Additional epidemiological studies, suchas family-based or day care studies, will be useful in clari-fying the role of HCV as a cause of human gastroenteritis inthe United States. It is of interest that one report from theUnited States (1) suggested that most of the 27-nm virusesisolated from patients with gastroenteritis were not Norwalkvirus or astrovirus; some of them could be caliciviruses.Recent studies (8, 14) indicated that antibodies to two

distinct strains of HCV are common in immunoglobulinpools from different countries (Japan, the United Kingdom,Canada, the United States, Belgium, Switzerland, SouthAfrica, Australia, Saudi Arabia, Singapore, Papua NewGuinea, and Indonesia). There is, however, little informationon the prevalence of HCV infection in the United States.Our serological survey indicates that infection with HCV isprevalent in the United States, Canada, and the People'sRepublic of China, where gastroenteritis due to HCV hasrarely or never been reported. Age-related prevalence ofantibody to HCV reported here was similar to that in Japanand the United Kingdom (8, 20) and indicated that infectionswith HCV begin in early infancy and are acquired by the ageof 6 years, whereas group A rotavirus infections are acquiredby the age of 3 years. The recent report by Cubitt andMcSwiggan (8) in which the vast majority of sporadic casesof HCV infection in the United Kingdom were found amonginfants aged between 3 months and 5 years agrees with ourfindings. The high prevalence of antibody detected in serumsamples obtained from infants below the age of 3 months,which declined to 25% in infants aged 4 to 11 months, alsosuggests that the antibody found in early infancy was prob-ably passively acquired from mothers. It remains unknownwhether such naturally derived antibodies mediate protec-tion from infection in young children (8, 13). The conversionof existing RIA to the ELISA and ELISA-BL for HCV andfor antibodies to HCV should facilitate further studies oftheir role as causes of viral gastroenteritis.

ACKNOWLEDGMENTS

This work was supported by the Baylor Virus GastrointestinalResearch Fund and by a grant from the Health Care Division ofRichardson-Vicks, Shelton, Conn.We thank John Burns and Margaret Conner, who helped produce

the mouse and rabbit antisera, and our colleagues who contributedvirus or serum samples for this work. We also thank David Matsonfor discussions of this work and the manuscript.

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Yolken, J. O. Arrobio, A. Z. Kapikian, R. H. Parrott, and R. M.Chanock. 1981. Comparison of direct electron microscopy,immune electron microscopy, and rotavirus enzyme-linked im-munosorbent assay for detection of gastroenteritis viruses inchildren. J. Clin. Microbiol. 13:976-981.

2. Chiba, S., S. Nakata, I. Nakamura, K. Taniguchi, S. Urasawa,K. Fujinaga, and T. Nakao. 1983. Outbreak of infantile gastroen-teritis due to type 40 adenovirus. Lancet li:954-957.

3. Chiba, S., Y. Sakuma, R. Kogasaka, M. Akihara, K. Horino, T.Nakao, and S. Fukui. 1979. An outbreak of gastroenteritisassociated with calicivirus in an infant home. J. Med. Virol. 4:

249-254.4. Chiba, S., Y. Sakuma, R. Kogasaka, M. Akihara, H. Terashima,

K. Horino, and T. Nakao. 1980. Fecal shedding of virus inrelation to the days of illness in infantile gastroenteritis due tocalicivirus. J. Infect. Dis. 142:247-249.

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6. Cubitt, W. D., and A. D. T. Barrett. 1984. Propagation of humancandidate calicivirus in cell culture. J. Gen. Virol. 65:1123-1126.

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8. Cubitt, W. D., and D. A. McSwiggan. 1987. Seroepidemiologicalsurvey of the prevalence of antibodies to a strain of humancalicivirus. J. Med. Virol. 21:361-368.

9. Cubitt, W. D., D. A. McSwiggan, and S. Arstali. 1980. Anoutbreak of calicivirus infection in a mother and baby unit. J.Clin. Pathol. 33:1095-1098.

10. Cubitt, W. D., D. A. McSwiggan, and W. Moore. 1979. Wintervomiting disease caused by calicivirus. J. Clin. Pathol. 32:786-793.

11. Cubitt, W. D., P. J. Pead, and A. A. Saeed. 1981. A newserotype of calicivirus associated with an outbreak of gastroen-teritis in a residential home for elderly. J. Clin. Pathol. 34:924-926.

12. Dimitrov, D. H., D. Y. Graham, J. Lopez, G. Muchinik, G.Velasco, W. A. Stenback, and M. K. Estes. 1984. RNA electro-pherotypes of human rotaviruses from North and South Amer-ica. Bull. W.H.O. 62:321-329.

13. Nakata, S. 1986. Development of microtiter solid-phase radio-immunoassay for detection of human calicivirus antigens andantibodies, and its application for serovirological studies ofcalicivirus infection. Sapporo Med. J. 55:497-512.

14. Nakata, S., S. Chiba, H. Terashima, and T. Nakao. 1985.Prevalence of antibody to human calicivirus in Japan andSoutheast Asia determined by radioimmunoassay. J. Clin. Mi-crobiol. 22:519-521.

15. Nakata, S., S. Chiba, H. Terashima, Y. Sakuma, R. Kogasaka,and T. Nakao. 1983. Microtiter solid-phase radioimmunoassayfor detection of human calicivirus in stools. J. Clin. Microbiol.17:198-201.

16. Nakata, S., S. Chiba, H. Terashima, T. Yokoyama, and T.Nakao. 1985. Humoral immunity in infants with gastroenteritiscaused by human calicivirus. J. Infect. Dis. 152:274-279.

17. Nakata, S., M. K. Estes, D. Y. Graham, R. Loose, T. Hung, S.Wang, L. J. Saif, and J. L. Melnick. 1986. Antigenic character-ization and ELISA detection of adult diarrhea rotavirus. J.Infect. Dis. 154:448-455.

18. Nakata, S., M. K. Estes, D. Y. Graham, S. Wang, G. W. Gary,and J. L. Melnick. 1987. Detection of antibody to group B adultdiarrhea rotaviruses in humans. J. Clin. Microbiol. 25:812-818.

19. Riepenhoff-Talty, M., L. J. Saif, H. J. Barrett, H. Suzuki, andP. L. Ogra. 1983. Potential spectrum of etiological agents ofviral enteritis in hospitalized infants. J. Clin. Microbiol. 17:352-356.

20. Sakuma, Y., S. Chiba, R. Kogasaka, H. Terashima, K. Horino,and T. Nakao. 1981. Prevalence of antibody to human calicivirusin general population of northern Japan. J. Med. Virol. 7:221-225.

21. Suzuki, H., T. Konno, T. Kutsuzawa, A. Imai, F. Tazawa, N.Ishida, N. Katsushima, and M. Sakamoto. 1979. The occurrenceof calicivirus in infants with acute gastroenteritis. J. Med. Virol.4:321-326.

22. Terashima, H., S. Chiba, Y. Sakuma, R. Kogasaka, S. Nakata,R. Minami, K. Horino, and T. Nakao. 1983. The polypeptide ofa human calicivirus. Arch. Virol. 78:1-7.

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