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CLINICAL MICROBIOLOGY REVIEWS, Jan. 1989, p. 51-89 Vol. 2, No. 10893-8512/89/010051-39$02.00/0Copyright A) 1989, American Society for Microbiology

Human Viral GastroenteritisMARY L. CHRISTENSEN

Department of Pathology, Northwestern University Medical School, Chicago, Illinois 60611, and Virology Laboratory,Department ofPathology, The Children's Memorial Hospital, Chicago, Illinois 60614*

INTRODUCTION................................... 51ROTAVIRUSES................................... 52

Introduction ................................... 52Molecular Biology and Classification................................... 52Epidemiology................................... 53Clinical Features and Pathogenesis ................................... 56

Clinical features ................................... 56Pathogenesis................................... 56

Laboratory Methods and Diagnosis ................................... 57Cultivation ................................... 57EM................................... 58EIAs and LA tests ................................... 58Other detection methods ................................... 61Antibody detection................................... 61

Immunology ................................... 61Prevention................................... 65

Vaccines ................................... 65Chemical disinfection................................... 66

Treatment................................... 67ADENOVIRUSES................................... 68

Introduction ................................... 68Molecular Biology and Classification................................... 68Epidemiology................................... 68Clinical Features ................................... 70Laboratory Diagnosis ................................... 71Immunology ................................... 71Prevention and Treatment ................................... 72

NORWALK AND NORWALK-LIKE VIRUSES ................................... 72Introduction ................................... 72Physical Characteristics ................................... 72Epidemiology................................... 72Clinical Features and Pathogenesis ................................... 73

Clinical features...............................................................................................................73Pathogenesis................................... 73

Laboratory Diagnosis ................................... 74Cell and organ culture................................... 74In vitro assays................................... 74

Immunology ................................... 75Prevention and treatment................................... 75

CALICIVIRUSES................................... 76ASTROVIRUSES ................................... 77CORONAVIRUSES ................................... 77ACKNOWLEDGMENTS ................................... 78LITERATURE CITED ................................... 78

INTRODUCTION and Norwalk-like viruses (6, 87, 187, 278, 401), caliciviruses(112, 231), astroviruses, (229, 230), and possibly coronavi-

Until 15 years ago, the causes of acute nonbacterial ruses (322). The common enteroviruses are associated withgastroenteritis were unknown. However, during the 1970s, a relatively few cases of viral gastroenteritis (15). The variousnumber of viruses associated with this clinical syndrome viral agents were discovered by the method of electronwere discovered, and their presence in the stools of patients microscopy (EM), using EM to examine stools or intestinalwith gastroenteritis were eventually correlated with the biopsies from these patients (18, 33, 110, 253). As a group,disease process. These various viruses include rotaviruses these viruses are fastidious and cannot be cultivated in(18, 109), fastidious fecal adenoviruses (77, 110), Norwalk routine cell culture (22, 53, 70, 77, 84, 110). Some can now be

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propagated by using special cell types or techniques or both(320, 353, 372). In addition, a variety of in vitro detectionsystems, including enzyme immunoassays (149, 175, 333,408), radioimmunoassays (137, 141, 181, 264), latex aggluti-nation (140, 169), and nucleic acid hybridization techniques(116, 175), have been developed for their rapid detection.The detection and identification of these agents are impor-

tant since viral gastroenteritis is the second most commonclinical entity in developed countries, second only to viralupper respiratory tract illness (188). Worldwide, acute gas-troenteritis and its associated dehydration afflicts almost 500million children annually. In underdeveloped or developingcountries, acute gastroenteritis, including viral gastroente-ritis, is the leading cause of death of children under the ageof 4 years (360).

Since 1980, a wealth of literature has developed coveringmany aspects of these viruses and the infections they cause,including their molecular biology, epidemiology, immunol-ogy, and clinical features. In addition, detection tests forroutine use have been developed for rotaviruses (310) andfecal adenoviruses (333) and experimental rotavirus vaccineshave been developed (184, 377, 379, 380). Of these viruses,rotaviruses are the most common known cause of viralgastroenteritis in infants and young children, with the fastid-ious fecal adenoviruses most likely being the second mostcommon cause in this age group (301, 369). Calici-, astro-,and coronaviruses are probably responsible for a minority ofillness in the young age group (253, 302). In contrast, theNorwalk and Norwalk-like viruses have caused considerablenumbers of outbreaks of gastroenteritis among older chil-dren, adolescents, and adults (136, 137, 189).

ROTAVIRUSES

Introduction

Rotaviruses are in the Reoviridae family, the members ofwhich possess a double-layer of icosahedral shells of approx-imately 70 nm in diameter, with a core of double-strandedribonucleic acid (dsRNA). Rotaviruses infect a wide varietyof mammals and birds as well as humans. Most rotavirusesfrom various species are similar and share a common groupantigen, which is associated with the inner capsid layer.However, some rotaviruses do not possess this group anti-gen. Rotaviruses are responsible for a significant proportionof gastroenteritis in small children and infants, as well as forcausing gastroenteritis in the elderly. Similarly, animal andavian rotaviruses are responsible for gastroenteritis in theyoung of their corresponding species.

Molecular Biology and ClassificationRotaviruses contain a dsRNA genome consisting of 11

segments, ranging in molecular weight from 0.4 x 106 to 2.0x 106 (275). These 11 segments can be separated by poly-acrylamide gel electrophoresis (PAGE) (103, 182), using themethod of Laemmli (212). Different rotavirus isolates fre-quently exhibit differences in the electrophoretic mobilitiesof their 11 segments. Rotaviruses exhibiting these differentelectrophoretic mobilities have been termed electrophe-rotypes. Although frequently many of the rotaviruses iso-lated in the same geographical location at the same time ofyear may exhibit similar or identical electrophoretic mobili-ties, there has been considerable variation in electrophoreticmobilities of viruses isolated during different seasons or atdifferent locales during the same season. For example,

Rodger et al. (304) described 19 different electropherotypesisolated in Melbourne, Australia, from 1973 to 1979; Albertet al. (3) described 9 different electropherotypes isolated inIndonesia in 1978 and 1979; Rodriquez et al. (306) reportedon 10 different electropherotypes seen in two nosocomialoutbreaks for a 6-month period in 1979 and 1980 in Wash-ington, D.C.; and Spencer et al. (340) described 32 differentelectropherotypes seen in Santiago, Chile, from 1979 to1981. Attempts have been made to characterize and classifyhuman rotaviruses based on their electrophoretic mobilities.However, the variation has been too great, except for twopatterns of electrophoretic mobility that have been distin-guished (103, 182). These are the "short" pattern and the"long" pattern, in which gene segments 10 and 11 migratemore slowly, creating a short pattern, or more rapidly,creating a long pattern, in the polyacrylamide gels (103, 182).To study the problem of genetic relatedness among variouselectropherotypes, Flores et al. (115) prepared single-stranded RNA probes for hybridization to gene segmentswhich consist of dsRNA. They found that correspondinggene segments that exhibited similar migration patterns didnot necessarily exhibit RNA homology when studied byhybridization with the probes. Conversely, some corre-sponding gene segments that exhibited RNA homology withthe probes did not have the same electrophoretic migrationpattern. Thus, they concluded that similarities or differencesin electrophoretic mobility did not always reflect similaritiesor differences in genetic relatedness between various RNAsegments (115). However, it has been shown that electro-pherotypes are excellent markers for identifying and follow-ing the spread of viruses from one individual to another indiscrete outbreaks; thus, they are good for providing epide-miological information (47, 304).

In addition to segments 10 and 11, three gene segments areof particular interest since they code for three major rota-viral antigens, VP4, VP6, and VP7. The major antigens ofrotavirus and the genes that code for them are shown inTable 1. Gene segment 4 codes for VP4 (179, 219, 222, 243,335). Originally the product of gene 4 was called VP3, butLiu et al. (219) recently proposed that the gene 4 product becalled VP4 while the product of gene 3 be called VP3. Genesegment 6 codes for VP6 (180). Depending on the strain ofrotavirus, gene segment 8 or 9 codes for VP7 (25, 180). Forexample, gene segment 8 codes for VP7 in the humanserotype 2 rotavirus Hu/5 isolated in Melbourne, Australia(95), and for the bovine rotavirus UK (207). Gene segment 9codes for VP7 in the human serotype 1 rotavirus Wa and forthe simian rotavirus SA11 (180, 183, 207).VP4 is an outer capsid protein which has an apparent

molecular weight of 88,000 (241). VP4 is associated with twobiological functions involved with virus-cell interaction:hemagglutination and protease-enhanced plaque formation(179, 222, 241, 335). VP4 is also responsible for the restric-tion of growth in cell culture (114, 180).VP6 is the major inner core structural protein which is

present in large amounts in the virion. It has an apparentmolecular weight of 42,000 (25, 93, 94, 240, 275). VP6 is themajor subgroup antigen which can specify one of tworotavirus subgroups (subgroups I and II) (25, 180). Thesesubgroups have been differentiated on the basis of a varietyof tests. These include differentiation by electrophoreticmigration patterns of gene segments 10 and 11 in PAGE(104), the complement fixation (CF) test (420), the immuneadherence hemagglutination test, immune electron micros-copy (IEM) (420), the radioimmunoassay (RIA) (181, 249),and the enzyme-linked immunosorbent assay (EIA) (357,

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TABLE 1. Gene code assignments for rotaviral proteins

Gene Protein gene Approximate mol Location Biologic functions of VPsegment segment wt of protein

1 VP1 125,000 Core2 VP2 94,000 Core3 VP3 88,000 Core4 VP4 88,000 Outer capsid Hemagglutination; protease-enhanced

plaque formation; restriction of growthin cell culture

5 NS53 53,0006 VP6 41,000-42,000 Inner capsid Major inner core structural protein; major

subgroup antigen7 NS34 34,0008 or 9 VP7 34,000-40,000 Major outer capsid Serotype specificity; major neutralization

protein (glycosylated) determinant10 NS28 28,00011 VP9 26,000

a VP, Viral protein; NS, nonstructural protein.

418, 421), using either polyclonal sera or monoclonal anti-bodies (132, 355). However, some monoclonal antibodiesreact with viruses in both subgroups, so VP6 proteins mustalso share common epitopes.VP7 is the major outer capsid protein which is glycosyla-

ted and has an approximate molecular weight of 34,000 to40,000 (93, 106, 163, 180, 183, 207, 275, 335, 350). Thisprotein is responsible for the serotype specificity of the virus(10, 92, 94, 95, 106, 163, 180, 337), which was initiallydetermined by virus neutralization (166, 402, 404). AlthoughVP7 is the major determinant of neutralization, VP4 can alsoelicit neutralizing antibody (10, 135, 165, 272, 335). At leastfour human serotypes have been identified: serotypes 1, 2, 3,and 4 (106, 163, 404). Subgroup I includes human rotavirusstrains of serotype 2, which is represented by prototypestrain DS-1 (106, 357, 402, 404). Subgroup II includesserotype 1 (represented by prototype strain Wa) and se-

rotypes 3 and 4 (106, 165, 357, 371, 402, 404).Although at least four human serotypes have been identi-

fied (106, 163), a possible fifth serotype from Indonesia hasbeen described by Matsumo et al. (242). This virus strain,strain 69M, has a "super-short" RNA electrophoretic pat-tern of gene segments 10 and 11. By RNA-RNA hybridiza-tion, 69M was found to have a low degree of homology withthe representative strains of all four known human se-rotypes, and it could not be classified by neutralizationanalysis into any of these four serotypes (242). Albert et al.(4) also detected two similar super-short strains designatedB37 and B38 in Indonesia.A sixth possible serotype, strain W161 isolated in the

United States, has been described by Clark et al. (61). Bycross-neutralization tests, this virus was distinguished fromhuman rotavirus serotypes 1, 2, 3, and 4, from human strain69M, and from bovine (NCDV), porcine (OSU), and chicken(Ch2) rotaviruses (61).

Until recently it was thought that all human and animalstrains of rotavirus possessed a common group antigen (399).However, various rotaviruses have been isolated from hu-mans and animals that do not possess that common group

antigen originally reported by Flewett and Woode (113, 399).Rotavirus strains lacking the common group antigen havebeen isolated from humans, cows, lambs, pigs, rats, andbirds (34, 73, 81, 105, 124, 270, 305). These more newlydiscovered viruses are morphologically indistinguishablefrom other rotaviruses in that they consist of a double-shelled icosahedran containing 11 genome segments of

dsRNA; however, they have an electrophoretic pattern thatdiffers from that of known rotaviruses. These agents havebeen called "rotavirus like," (170, 305) "antigenically dis-tinct rotaviruses," (81, 101) "pararotaviruses," (24, 105,270) "atypical" rotaviruses (285, 286), and "novel" rotavi-ruses (171). More recently, Pedley et al. (285), studyingatypical porcine rotaviruses, introduced the designationgroup A, B, C, etc., analogous to influenza virus terminol-ogy. They proposed the usage of group A for the originalconventional rotaviruses with the common group antigenand groups B and C for more recently discovered atypicalrotaviruses that (i) possess other group antigens and (ii) aregenetically different based on (a) electrophoresis of the 11gene segments and (b) one-dimensional terminal finger-printing analysis of the RNA segments. Later, Pedley et al.(286), after analyzing atypical porcine and chicken rotavi-ruses, described group D and E rotaviruses.Human rotaviruses belonging to groups B and C have now

been described. Atypical strains were analyzed by antigenicanalysis (IEM, immunofluorescent-antibody assay, and/orEIA) and by genome profile analysis, terminal fingerprintanalysis of genome segments, and/or dot blot hybridization(34). Sporadic single observations of group C rotaviruses inhumans have been made in a few laboratories (34). Forexample, analysis of two atypical rotaviruses from Australiaand Brazil were found to be in group C (34). Eiden et al.(101), found that five of six human isolates from the UnitedStates were related to porcine and bovine group B rotavi-ruses. In China, group B rotaviruses have been found tocause severe epidemics of diarrheal disease (48, 170, 171).An unusual characteristic of the outbreaks in China was thata much higher attack rate was noted among adults thanamong children. However, Dai et al. (73) described anoutbreak of diarrhea among newborns caused by the newChinese rotavirus that was placed in group B by Chen et al.(48). This is probably the first report of neonatal infectioncaused by group B virus since group A rotaviruses have beenthe primary cause of outbreaks in neonatal nurseries in anumber of countries. To summarize, rotaviruses can beclassified by four main categories: group specificity, sero-type specificity, subgroup specificity, and strain specificity.

Epidemiology

Rotaviruses were first discovered in humans 15 years agoby Bishop et al. (18, 19) by the EM examination of duodenal

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FIG. 1. Common gastroenteritis viruses from stool specimens of pediatric patients with acute gastroenteritis. For each, an approximately20% stool suspension was prepared in 1% ammonium acetate and negatively stained with 2% phosphotungstic acid; x 140,000. (A)Rotaviruses; (B) adenoviruses in stool; (C) caliciviruses; (D) astroviruses. Electron photomicrographs courtesy of Cynthia Howard, TheChildren's Memorial Hospital, Chicago, Ill.

mucosal epithelial cells obtained by duodenal biopsy of agroup of Australian children hospitalized with nonbacterialgastroenteritis. In the same month, Flewett et al. (109)described similar reoviruslike particles seen by EM in stoolspecimens from patients with gastroenteritis in the UnitedKingdom. Shortly thereafter, the finding of these viruses inpatients, but not controls, was reported from Singapore(354), Canada (252), southern Africa (65), India (164), theUnited States (186), and Scandinavia (277). Flewett et al.(111) suggested the name "rotavirus" based on the wheel-like appearance (Fig. 1A) of these viruses.The seriousness of rotavirus infection was exemplified by

the reports of Middleton et al. of seven fatal cases occurringout of 60 rotavirus-infected patients (252) and a review of 21fatal cases (41). Others reported that this virus was alsoresponsible for nosocomial outbreaks of gastroenteritis innurseries (40, 56, 261). Chrystie et al. (56) also noted that inneonates the disease was mild or symptoms were lackingentirely. In addition, young adults, i.e., parents of rotavirus-infected infants, infrequently excreted large amounts ofvirus and infrequently developed symptoms or had only mildsymptoms (56, 185, 261, 307). Often the only evidence ofinfection in parents was determined by fourfold or greaterrises in serum antibody titers (185).

Rotaviruses are responsible for approximately 50% ormore of the gastroenteritis in hospitalized pediatric patientsduring the cooler months of the year in parts of the worldthat have temperate climates (15, 76, 152, 185, 203). Theseworldwide reports of the high incidence of rotavirus infec-tion have come from urban populations. However, theincidence of rotavirus infection appears to be somewhatlower or absent in patients from rural areas as reported fromAustralia and South Africa (89, 321-323). There may beseveral reasons for this. One is that rotaviral colonization ofneonates may lead to protection against symptomatic infec-tion later in infancy. Another reason may be that, in someinstances, a lack of overcrowding may prevent the virusfrom spreading easily. Also, differences in diet or lifestyle incertain rural areas may contribute to a different viral gutflora.

Since many reports indicate that rotavirus infection in-creases during the cooler months, the role of weather inaltering the rotavirus infection rate has been studied invarious parts of the world by a number of investigators. Intemperate climates such as in northern Japan, increases inthe infection rate appeared to be related to the drop intemperature, but not to the outside relative humidity (205).In Washington, D.C., rotavirus gastroenteritis increased


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after a month of cold or dry weather rather than warm or wetweather (28). Brandt et al. (28) suggested that indoor crowd-ing and low indoor relative humidity may increase aerosoli-zation of rotavirus particles on surfaces, as well as contrib-ute to dehydration of infected infants during these months.However, in another report, rotavirus prevalence in south-ern California during one season (October to December) didnot correlate with either the coldest or the driest months ofthe year. In some tropical countries, including Nigeria (282),southern India (235), Bangladesh (20), Indonesia (336), Ec-uador (348), and Costa Rica (152), rotavirus infections tendto peak during the dry season. However, no distinct seasonalvariation occurred in other reports from tropical countries,including Ecuador (345) and Venezuela (80) and, in certainreports, South Africa (323). Thus dryness may have someeffect on the spread of the virus, although in some tropicalcountries any variation in temperature and humidity may betoo slight to be of any significance.Murphy et al. (261) found no seasonal variation in noso-

comial neonatal nursery infection in Sydney, Australia.However, indoor temperature and humidity may not havevaried due to environmental control, and once rotaviralcolonization of a nursery occurs, it may be difficult toeradicate. Dennehy and Peter (78) found that three-fourthsof the nosocomial infections they studied occurred in thewinter rotavirus season. Hjelt et al. (159) found that approx-imately half of the nosocomial rotavirus infections theyinvestigated occurred during an epidemic outbreak whereasthe other half were scattered with regard to season. A majorrisk factor was the duration of patient stay. Nosocomialinfections appeared to be due to failure to isolate thenosocomially infected patients, although the community-infected patients were put in private rooms upon admission.Sharing of nursing and medical staff did not differ amongcontrol patients, although this has been a factor in somestudies. In one study (156), 36% of the nursing staff in thepediatric wards were infected with rotavirus as comparedwith 15% of the nursing staff in other wards. Asymptomaticor mild symptoms were characteristic of staff rotavirusinfection.Asymptomatic rotavirus infection and viral carriage occur

frequently and have been studied by several investigators.Champsaur et al. (46) found rotavirus in 36% of hospitalizedchildren with diarrhea and in 24% of hospitalized childrenwithout diarrhea. Of those children shedding rotavirus, 48%were not diarrheic. Virus shedding that was not associatedwith diarrhea was observed in 71% of neonates, 50% of 1 to6 month olds, and 26% of 7 to 24 month olds. In a similarstudy (45) of patients ranging in age from newborns to 24months admitted to the hospital, Champsaur's group foundthat 13% had symptomatic disease (diarrhea and serologicresponse), 7% had asymptomatic infection (no diarrhea, butserologic response), and 20% were viral carriers (no diarrheaand no serologic response). Asymptomatic infection as de-fined by serologic response but no diarrhea occurred in 2%of neonates, 20% of 1 to 6 month olds, and 37% of 7 to 24month olds. Virus carriage as defined by no diarrhea and noserologic response occurred in 27, 19, and 14% of thesechildren, respectively.Walther et al. (389) studied 871 children admitted to a

pediatric ward for various conditions. Of 742 asymptomaticchildren, 38% were excreting rotavirus as determined byEIA. Of 129 symptomatic children, 50% excreted rotavirus,26% excreted enteric pathogens, and 13% excreted bothrotavirus and enteric pathogens, and in 37% no agent wasfound. Thus, the presence of rotavirus has not always

correlated with disease. Rotavirus is also prevalent in day-care centers and can be spread to family contacts, thuspropagating the infection in the community (191). Manyday-care children are asymptomatic, indicating a large res-ervoir of infection. The numbers of infected children rangefrom 10 to 30%, depending on the study (9, 191). In onestudy (9) 20% of the adults directly involved in child carewere infected, although they were primarily asymptomatic.However, adults have also experienced more severe clin-

ically acute cases of rotavirus gastroenteritis. von Bonsdorffet al. (385) reported acute rotavirus gastroenteritis in 45% ofadult hospital employees aged 19 to 62, and Echeverria et al.(98) reported rotavirus in 5% of adult patients aged 16 to 72admitted to the hospital for gastroenteritis.

Rotavirus gastroenteritis has been more severe in theelderly, according to reports from nursing homes and otherinstitutions for the elderly (66). Halvorsrud and Orstavik(142) reported 92 cases of mild-to-severe, rotavirus-associ-ated gastroenteritis in 70 to 90 year olds in a nursing home.Initial symptoms of nausea and vomiting were followed bydiarrhea and low-grade fever. One patient died. There ap-peared to be a great susceptibility to both infection anddisease, since 66% of the patients in the affected wardsbecame ill. Similarly, a serious rotavirus outbreak occurredin 56% (19 of 34) of patients in a hospital geriatric ward, aswell as in staff members (238). Six patients had severe illnessand two died. Forty percent of asymptomatic geriatricpatients were infected. Outbreaks among the elderly canbecome extensive and severe, possibly because of loweredimmunity. Thus, in this population, testing of all staff andpatients is important to identify all infected individuals andto institute control measures.Although group A rotaviruses have been responsible for

almost all of the rotavirus infections in both the young andthe elderly in North America and Europe, a new group ofrotaviruses has been responsible for large outbreaks ofsevere rotavirus diarrhea occurring in adults of all ages inChina (171). These rotaviruses did not share the groupantigen (group A) of known rotaviruses at the time and werefound to belong to a new group, group B (48). Humaninfection with group B rotavirus has not been widespreadoutside of China, however (267, 368).

Within the group B rotaviruses, subgroups and types havenot been delineated as has been done with the group Arotavirus. Within the A group of rotaviruses, the prevalenceof the subgroup and the type of rotavirus causing infectionhave been studied. White et al. (394) subgrouped 99% of 252specimens obtained from Venezuelan children over a 45-month period. Some 14% shed subgroup I and 85% shedsubgroup II rotavirus. Of the subgroup I viruses, one-halfwere shed during a 3-month period. There was no differencein the occurrence of fever and vomiting between the childrenwith either subgroup, but patients with subgroup II infec-tions had longer-lasting illness.Yolken et al. (418) studied 414 rotaviruses isolated from

patients in Washington, D.C., Belgium, and Central Amer-ica. They found that 23% were type 2 (subgroup I) and 77%were type 1 (subgroup II), with a similar distribution fromvarious parts of the world. In an analysis of children whowere reinfected, sequential infections usually involved dif-ferent serotypes, and illness caused by one serotype did notprovide resistance to illness caused by the other serotype.Studying the prevalence of various subgroups and types is animportant consideration for determining which rotavirustype(s) to incorporate into rotavirus vaccines.

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Clinical Features and Pathogenesis

Clinical features. Several groups have studied the clinicalcharacteristics of rotavirus infection, in particular, in hospi-talized patients (161, 367, 381). The two most prominentfeatures were vomiting and diarrhea, usually with suddenonset. Vomiting appeared to be the most common firstsymptom, occurring first in 34 to 55% of patients, dependingon the study. Vomiting often preceded the onset of diarrheaby a few hours to 24 h and occurred at some time duringillness in 48 to 92% of patients, with a mean duration of 3days. Diarrhea was the first symptom to occur in 23 to 29%of patients, depending on the study. Diarrhea occurred atsome time during illness in 65 to 100% of patients and lasteda mean of 5.0 to 5.9 days. Vomiting and diarrhea startedsimultaneously as the first symptoms in 22% of patients.Characteristically, there was an absence of blood in thestools (161). Fever occurred first in 13% of patients andoccurred at some time during illness in 34 to 86% of patients.Abdominal pain was somewhat infrequent, occurring in 17 to29% of patients. Respiratory symptoms occurred first in 24%of patients and occurred at some time during the illness in 24to 52% of patients. Overall, symptoms were more severe inhospitalized patients than in patients not requiring hospital-ization, and patients in poorer general condition were moredehydrated.There have been conflicting reports on the role of rota-

virus in causing respiratory infection and symptoms. Lewiset al. (216) reported respiratory illness in 66% of rotavirusgastroenteritis patients versus 26 to 38% of nonrotavirusgastroenteritis patients. Respiratory symptoms usually pre-ceded gastrointestinal symptoms, although they also oc-curred concurrently, and consisted of rhinitis, pharyngitis,tonsillitis, and otitis media.Yolken and Murphy (413) identified rotavirus by EIA in

tracheal aspirates from two of five infants who died ofsudden infant death syndrome who had acute upper respira-tory infection. Although none of these infants had gastroen-teritis, rotavirus was also detected in their stools. Similarly,Santosham et al. (317) detected rotavirus in respiratorysecretions from 4 of 45 pediatric patients with pneumonia.None of the four had diarrhea. However, since they hadprior antibiotic treatment, and Mycoplasma sp. was alsofound in one child, bacteria may have been the cause of thepneumonia. Fragoso et al. (118) also detected rotavirusantigen in respiratory secretions in 2 of 30 children with bothupper respiratory tract infection and vomiting.To study the role of rotavirus in respiratory infection,

Prince et al. (291) used a murine rotavirus model to demon-strate that aerosol transmission can occur. The result wasboth respiratory and gastrointestinal infection and gastroen-teritis. The authors concluded that, since rotavirus may befound in respiratory secretions of children, it may be trans-mitted by the respiratory route as well as the fecal-oralroute.However, several groups have failed to show any signifi-

cant role of rotaviruses in respiratory infection. Goldwater etal. (127), using direct EM, IEM, and EIA, failed to detectrotavirus in respiratory secretions of rotavirus-infected pa-tients. Their study included specimens obtained from pa-tients with respiratory symptoms prior to the onset ofgastroenteritis. Similarly, Vollet et al. (384) could not detectrotavirus in 11 of 13 children with rotavirus diarrhea whoalso had respiratory symptoms.Maki (236) found no difference in respiratory symptoms in

rotavirus gastroenteritis patients (53%) or nonrotavirus gas-

troenteritis patients (62%). Hjelt et al. (161) found no moreupper respiratory tract symptoms in rotavirus gastroenteritispatients (36%) than in nonrotavirus gastroenteritis patients(35%). Similarly, Uhnoo et al. (367) observed respiratorysymptoms in 32% of rotavirus gastroenteritis patients, butthese data were not significant when compared with respira-tory symptoms in patients with gastroenteritis caused byother viruses or bacteria or when no agent was detected.These data suggest that rotaviruses may sometimes cause

respiratory symptoms. Differences seen in the various re-ports may depend on the locale, the year, and the strains ofrotaviruses involved. Some patients with nonrotavirus gas-troenteritis and respiratory symptoms had infection causedby adenoviruses, which are known to cause both gastroin-testinal and respiratory symptoms simultaneously (seebelow). Thus, the respiratory symptoms occurring in theadenovirus gastroenteritis groups may have contributed tothe lack of significance observed between the rotavirus andnonrotavirus gastroenteritis groups in some of the reports.

Various other complications have been reported to occurwith rotavirus infection. Fernbach and Lloyd-Still (107)reported three patients with severe, prolonged rotaviruscolitis with bloody stools. Wong et al. (397) reported a caseof aseptic meningitis associated with rotavirus gastroenter-itis in which rotavirus particles were seen in the cerebralspinal fluid by IEM. Ushijima et al. (373) reported on a caseof encephalitis that developed during rotavirus gastroenter-itis, in which rotavirus immunoglobulin G (IgG) increased inthe patient's cerebrospinal fluid. In addition, rotavirus-likeparticles have been detected by EM and EIA in a liverbiopsy in a case of hepatic abscess (139).

Pathogenesis. Rotavirus infection is spread primarily bythe fecal-oral route. Although rotavirus is relatively acidlabile, rotavirus can survive the pH of a stomach that isbuffered, or can survive in the stomach after a meal. At pH2.0 (that of a fasting stomach), rotavirus is rapidly inacti-vated in <1 min (393). However, at pH 3.0, inactivation ismuch slower, the viral half-life being about 10 min; at pH4.0, inactivation is minimal. The infant gastric pH tends to beapproximately 3.2, and in general, the stomach pH remainsabove 3.0 for at least 1 h after a meal. This probably explainsthe efficient transmission of rotavirus.To determine the infective dose required to produce

infection with or without symptoms, adult volunteers in-gested 0.009 to 90,000 focus-forming units of rotavirus (390).Results showed that as little as 0.9 focus-forming unit causedinfection in one of nine volunteers, as determined by viralshedding and a significant rise in antibody titer. A 9-focus-forming unit dosage caused infection in 8 of 11 volunteers,with 6 of these having symptoms. Higher viral doses causedhigher percentages of infection, but no increase in thenumber of individuals with symptoms.

Rotaviruses tend to infect the small intestine, as do othergastroenteritis viruses. In particular, rotavirus replicationtakes place in epithelial cells on the tips of villi of the smallintestine, and infection is confined primarily to these cells(405). Rotaviruses selectively infect the mature villus entero-cytes of the small intestine; rotaviruses exhibit a predilectionfor young animals of many animal species (273).The histology of duodenal biopsies obtained from rota-

virus gastroenteritis patients was first described by Bishop etal. (18). A patchy irregularity of the mucosal surface wasseen in most cases. Mucosal changes ranged from mild tosevere. The changes included shortening and blunting of villiand increased infiltration of the lamina propria with mono-nuclear cells. Epithelial cells were more cuboidal and less



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regular than usual. By EM, reovirus-like particles were seenin the epithelial cells of the duodenal mucosa. Virus particleswere seen within distended cisternae of the endoplasmicreticulum in vacuolated cells. No virus-like particles wereobserved in any of the other types of cells in the laminapropria.Davidson and Barnes (74) found mucosal damage to be

quite variable and often patchy. Mild changes in seven oftheir patients included broadening of villi, mild cellularinfiltration of the lamina propria, and early epithelial celldamage. Moderate changes in eight patients involved con-siderable blunting of villi, obvious increase in inflammatorycells in the lamina propria, increased crypt depth, andflattening of epithelial cells. Severe changes in two patientsshowed complete villous flattening, marked inflammatorycell infiltration, crypt hypertrophy, and severe epithelialdamage with cuboidal epithelium. The severe damage couldbe confused with the structural appearance seen in coeliacdisease. Rotavirus particles were observed by EM in thecytoplasm of infected cells. Mucosal damage was rapidlyrepaired, as early as 3 weeks after onset. They found thatchildren with a more severe mucosal lesion were more likelyto become dehydrated and require intravenous therapy forrehydration.

In bacterial gastroenteritis, abdominal pain, bloody stools,leukocytosis, and prolonged diarrhea are more likely tooccur than in viral gastroenteritis (367). Viral gastroenteritisalso differs from bacterial gastroenteritis in its physiology(360). In rotavirus infection, there is patchy replacement ofinfected mature villous tip cells by secretary crypt cells,decreased intracellular Na+,K+-adenosine triphosphataseactivity, and impairment of glucose-coupled sodium trans-port (360). Abnormally low maltase, sucrase, and lactaselevels were also found in children with rotavirus gastroen-teritis (18, 74), which returned to normal after 4 to 8 weeks.Most (but not all) children with acute rotavirus gastroenter-itis have lactose malabsorption and intolerance (172). Anincrease in diarrhea can occur after feeding lactose, so that anon-lactose-containing formula is usually given during rota-virus illness when infants are able to take fluids orally.Normal lactose tolerance reappears by at least 10 to 14 daysafter the start of illness, when lactose-containing productscan then be introduced (172).Loss of fluids and electrolytes in rotavirus gastroenteritis

can lead to severe dehydration and even death and requiresfluid and electrolyte replacement therapy. In developingcountries, recurrent bouts of gastroenteritis can lead to avicious cycle of protracted diarrhea, food intolerance, andmalnutrition (360). The malnutrition may be further com-pounded by frequent fasting, which is a measure commonlyused in the management of acute gastroenteritis in somedeveloping countries (360). For marginally nourished ormalnourished children, diarrhea with associated starving canhave deleterious effects. When a person has fasted for 3 to 5days, depletion of intestinal digestive enzymes and gut massoccurs and absorption of water, salt, glucose, disaccharides,and amino acids are substantially reduced (360).

In spite of these multiple physiological abnormalities, oralreplacement therapy is effective in correcting dehydration inrotavirus gastroenteritis. Its effectiveness is probably due tothe presence of intact glucose-coupled sodium transport innoninfected bowel (360).

Laboratory Methods and DiagnosisSince the discovery of rotaviruses by EM, a number of

rotavirus detection methods have been developed. Since

rotaviruses have been difficult to propagate in cell culture,other viral and antigen detection methods have been used.These are based primarily on antigen-antibody reactions.Probably the most commonly used procedures at the presenttime are EIA and latex agglutination (LA) tests, sinceseveral commercial kits are available for use in many coun-tries. EM procedures are also used. A variety of other viraland antigen detection tests have been developed, but areused primarily as research tools in the laboratories thatdeveloped them.

Cultivation. Propagation of human rotaviruses is usuallynot carried out in diagnostic laboratories since virus is foundin large quantities in stool specimens and can be rapidlydetected by antigen detection tests. However, some re-search laboratories have cultivated rotaviruses by usingvarious manipulations. Wyatt et al. (403) propagated a strainof human rotavirus (Wa strain, serotype 2) in primaryAfrican green monkey kidney cell cultures after virus frompooled human stools had been passed 11 times in newborngnotobiotic piglets. Sato et al. (319) and Urasawa et al. (372)reported the propagation of a number of human rotavirusesfrom stool specimens by using a combination of threetechniques. Using roller cultures of MA-104 cells, a line offetal rhesus monkey kidney cells, they added low levels oftrypsin to the maintenance medium and pretreated theirspecimens with trypsin. Each of these techniques had failedwhen used separately.

Similarly, Kutsuzawa et al. (211) used trypsin treatmentand roller cultures of MA-104 cells on two stool specimens,one of which contained a subgroup I rotavirus and the otherof which contained a subgroup II human rotavirus. Dis-tinctly recognizable cytopathic effect (CPE) was observedby passage 6 of the subgroup I isolate and by passage 3 of thesubgroup II isolate. CPE consisted of obscure cell bound-aries, cell fusion, cell rounding, cell detachment, and lyticfoci. Supernatant fluids were trypsin treated prior to eachpassage.Hasegawa et al. (146) also propagated a number of human

rotavirus isolates from stool samples, using trypsin pretreat-ment and trypsin in the maintenance medium. However,they found that rolled primary cynomolgus monkey kidneycells were more sensitive than the rolled MA-104 cells. CPEappeared at passage 2 to 7, although virus could be detectedin the supernatant fluids of passage 1 by the immune adher-ence hemagglutination test. Passaged fluids were not trypsintreated after the initial inoculation.

Birch et al. (16) studied nine strains of rotavirus in MA-104and CV-1 cells, a line of African green monkey kidney cells.In MA-104 cells, CPE consisted of a sloughing of cells, andin CV-1 cells, CPE consisted of an increased granularity ofthe cells. They found that CPE was not a reliable indicator ofreplication; CPE occasionally disappeared for severalpasses, although virus was detectable in the supernatantfluids by indirect fluorescent-antibody staining, EM, or EIA.Because of inapparent rotaviral CPE, Suzuki et al. (347)

described an interference test, similar to that used to detectrubella virus, to detect replicating rotavirus. Using twolaboratory adapted rotavirus strains of Kutsuzawa et al.(211) and the Wa strain, they found that, by challenging withcoxsackievirus B-1, interference could be detected 4 daysafter virus infection.Ward et al. (391) compared the growth of human rotavi-

ruses from stool specimens by using two types of primarymonkey kidney cell cultures (African green and cynomolgus)and two types of monkey cell lines (MA-104 and CV-1).Primary cells supported virus growth directly from the

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specimens much more effectively than the two continuouslines. Although viruses from the specimens could not alwaysbe grown in the cell lines, the viruses appeared to be fullyadapted for growth in the cell lines after two passes inprimary cells. The efficiency of viral growth also increasedwith cell passage. Only 1 of 46,000 virions in stool specimensinfected the primary cell cultures, whereas 1 of 6,600 visionswere infectious after three passes in primary cells.

Agliano et al. (1) reported the isolation of eight humanrotaviruses in LLC-MK2 cells (a continuous line of rhesusmonkey kidney) and human embryonic fibroblasts, using nospecial techniques. The cells were not rolled, and trypsinwas not incorporated into the maintenance medium nor usedto pretreat the stool specimens. They suggested that theserotaviruses may differ from other previously isolated rotavi-ruses, since special techniques were not needed to isolatethem. These cultivation methods, however, are not usedroutinely.EM. Initially, rotaviruses were detected directly in stool

samples by the EM of virus particles negatively stained withphosphotungstic acid, and this method is still used as thestandard (252, 253). A simple method consists of making anapproximately 20% suspension of the stool sample in dis-tilled water or 1% ammonium acetate. This suspension isthen placed on a Formvar-coated EM grid, and the excess isblotted. Phosphotungstic acid solution (2%) is added and theexcess is blotted. Phosphotungstic acid at pH 4.5 has beenshown to be optimal for both EM and IEM (268). It is anelectron-dense negative stain which does not stain the virusparticles, but rather the area around them, causing theunstained virions to stand out. Alternative negative stainsthat have been used are ammonium molybdate and uranylacetate (268). Since children usually have had several epi-sodes of diarrhea before their parents seek medical atten-tion, usually no bacteria are remaining in their stools by thetime a sample is submitted to the laboratory. However, somelaboratories prefer to clarify stool suspensions (20%) bylow-speed centrifugation. In some instances the clarifiedsupernatant is subjected to ultracentrifugation to pellet andconcentrate any virus particles present. The resuspendedpellet is then used for preparing grids, followed by negativestaining.

In IEM, patients' convalescent sera are mixed with a virussuspension. These suspensions can be obtained by purifyingvirions from stools or from infected cell culture fluids. Afterincubation of the virus-serum mixture, the mixture is placedon a grid and stained with phosphotungstic acid.

Nicolaieff et al. (269) described an IEM technique whichinvolved coating EM grids with Staphylococcus aureusprotein A, followed by adsorption of specific rotavirusantiserum to the protein A on the grid. They found that theirtechnique detected rotavirus particles in 3.5 times as manyspecimens as by routine EM. Svensson et al. (349) alsodescribed a technique almost identical to that of Nicolaieff,which they called solid-phase IEM (SPIEM). They foundtheir SPIEM to be 30 times more sensitive than routine EMor routine IEM and 10 times more sensitive than indirectEIA.Gerna et al. (121, 124) used SPIEM to distinguish between

human serotypes 1, 2, 3, and 4. S. aureus protein A was firstplaced on a Formvar-coated grid. Then type-specific, cross-absorbed polyclonal immune sera were adsorbed to theprotein A. Viral specimens were then added and observed byEM.

Various other modifications of EM have been reported.Kjeldsberg and Siebke (201) described a simple immunosor-

bent EM which allows one to wash specimens to removecontaminating material such as sucrose solutions from virussuspensions prior to negative staining of grids. Kjeldsberg(200) also described an EM technique for specific labeling ofhuman rotavirus with gold-IgG complexes.ETAs and LA tests. Since EM procedures are time-con-

suming to perform for a large number of samples, othertesting procedures were developed to detect rotaviruses orrotaviral antigens. The more universally used tests today arethe LA tests and the EIA. The EIA is based on principlesand procedures first described by Engvall and Perlmann(102) and Voller et al. (383). The first rotaviral ETAs weredescribed by Yolken et al. (408, 416) and others. EIAs aresimilar to RIAs, except that an enzyme is linked to thedetector antibody, instead of a radioisotope.Most of the EIAs utilize a three-layer double-antibody

sandwich technique (408). Briefly, anti-rotavirus hyperim-mune serum (or monoclonal antibodies) are adsorbed to asolid phase (13, 63, 162, 287, 349). This first antibody hasbeen termed the "capture," "coating," "catching," or"primary" antibody. Next the specimen is added, and ifvirus is present, it will bind to the rotavirus antibody. Asecond antibody, the "detector" or "secondary" antibody,is then added. In many EIAs, the second, detector antibodyis conjugated to an enzyme, making it also an "indicator"antibody. In some EIAs, however, the detector antibody isnot conjugated to an enzyme. In these instances, a third,"anti-antibody" or indicator antibody which is enzymeconjugated is added (144, 167, 318, 421). Often the thirdantibody has been used to obtain a more sensitive test or ithas been used for typing. This type of test is termed a"four-layer EIA" or an "indirect EIA."Various other modifications of the EIA procedure have

been described. Periera et al. (287) developed a four-layerrotavirus-adenovirus combination EIA. Either rotavirus oradenovirus antiserum as capture antiserum was added toalternate rows in a microtiter plate. After incubating thesamples, a single detector antibody was added, consisting ofa mixture of guinea pig (GP) antirotavirus and GP antiade-novirus sera. A third, indicator antibody, consisted of rabbitanti-GP conjugate.One major modification of the EIA has been the use of

typing sera or monoclonal antibodies for typing rotavirusesthat are detected in stool specimens. Although IEM can alsobe used to serotype rotaviruses (420), it is not practical forlarge numbers of specimens, and serotyping by the CFmethod is not as sensitive as other methods (418, 420, 421).

Zissis and Lambert (421) developed a type 1 and a type 2specific EIA, using type-specific antisera as both the captureantibody and the detector antibody, plus a third indicatorantibody-conjugate. Thouless et al. (357) also used poly-clonal serotyping reagents in an EIA to distinguish humanrotavirus serotypes 1, 2, and 3.

Singh et al. (332) and White et al. (394) developed an EIAby using monoclonal antibodies to either subgroup I or II,which reacted to the 42,000-dalton inner shell protein. Shawet al. (326) developed an EIA with monoclonal antibodies forserotypes 1 (human strain Wa) and simian serotype 2, whichreacted with the VP7 of each serotype.Coulson et al. (64) also reported on an EIA to type human

rotavirus, using neutralizing mouse monoclonal antibodiesspecific for serotypes 1, 2, 3, and 4 as detector reagents. Allof the monoclonal antisera, except one, were directed to themajor outer capsid protein gp34 (VP7), and one was directedto p84 (VP4, originally called VP3). The capture antibodieswere hyperimmune rabbit antiserum to human rotaviruses



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types 1, 2, 3, and 4. Previously, the rotavirus serotype hadbeen determined by cross-neutralization assays with hyper-immune antisera (64).A number of commercial EIA rotavirus detection tests are

also now available. Several of these have been extensivelyevaluated and reported on in the literature. These include theRotazyme I and Rotazyme IT EIA tests (Abbott Laborato-ries, North Chicago, Ill.), the Enzygnost EIA test (BehringInstitut, Marburg, Federal Republic of Germany), the Path-finder EIA test (Kallestad Laboratories, Austin, Tex.), andthe Bio-EnzaBead EIA test (Litton Bionetics, Charleston,S.C.).There are several reports comparing Rotazyme and Enzy-

gnost with EM and IEM. Yolken and Leister (410) evaluatedRotazyme I, Enzygnost, and indirect EIA and comparedthem with EM. They found the sensitivity of the indirectEIA, Rotazyme T, and Enzygnost to be 100, 93, and 88% andthe specificity to be 95, 95, and 89%, respectively.Cheung et al. (50) evaluated Rotazyme I versus EM. The

overall agreement was 88.7%, and the negative results had a91.95% agreement. The tests were read visually, and speci-mens with high Rotazyme readings correlated 100% withEM.Rubenstein and Miller (310) compared Rotazyme I with

EM and IEM. The levels of sensitivity were 106 particles perml for simian rotavirus SA11 and 107 particles per ml forhuman rotavirus. The sensitivity and specificity of RotazymeI compared with those of IEM were 98 and 92%, respec-tively. Rotazyme-positive specimens included those speci-mens that were EM negative but EIA positive that could beblocked in a blocking assay.Keswick et al. (190) also compared Rotazyme I and EM.

They found EIA to be more sensitive than EM and that theRotazyme test detected SA11 with a titer of 2 x 103plaque-forming units (PFU)/ml, which was a level of sensi-tivity greater than that reported by Rubenstein and Miller.They also carried out blocking assays on EM-negative andEIA-positive specimens and found the EIA-positive speci-mens to be true positives.Chernesky et al. (49) evaluated Rotazyme TT, a version of

Rotazyme with a shorter incubation time. They found that itwas 99.4% sensitive and 97.3% specific with an overallagreement of 98.7% when compared with EM on 229 sam-ples from patients aged 6 months to 6 years.

In addition to ETAs, LA tests have been developed byHaikala et al. (140) and Hughes et al. (169). Like EIA, anumber of LA tests are also commercially available and aresold under various brand names. One, Rotalex (OrionDiagnostica, Helsinki, Finland; sold in the United States byMedical Technology Corp., Somerset, N.J.) has been eval-uated and reported on by several groups of investigators. Itconsists of latex beads coated with anti-human strain Warabbit serum (330). Others include the Slidex Rota-Kit(Biomerieux, Marcy-l'Etoile, France).Doern et al. (83) compared 176 specimens in Rotazyme I,

Rotazyme TT, and Rotalex LA with a highly sensitive andspecific monoclonal antibody EIA, described by Herrmannet al. (151). They found the sensitivities of the Rotazyme Iand TT and LA to be 97.4, 100, and 81.6%, respectively, andthe specificities to be 88.8, 83.9, and 100%, respectively.Thus, the Rotazyme TT was more sensitive but less specificthan Rotazyme I or LA. Overall, Rotazyme I and TT werehighly sensitive, but both lacked specificity. They also noteda problem with a large number of specimens having equivo-cal results with Rotazyme I and TT.

Knisley et al. (202) used 100 specimens to evaluate four

tests and compare them with EM. The four tests were (i) theAbbott Rotazyme TT, a polyclonal antibody-based EIA; (ii)the Pathfinder EIA, a monoclonal antibody-based EIA; (iii) apolyclonal-based EIA, using reagents obtained from theNational Institutes of Health; and (iv) Rotalex LA. Thesensitivities were 73, 95, 57, and 61%, respectively, whilethe specificities were 88, 95, 96, and 98%, respectively.Gerna et al. (125) used 151 specimens and compared thePathfinder monoclonal antibody EIA with SPIEM as areference test. They found Pathfinder to have a sensitivity of98.7% and a specificity of 98.5%.

Cevenini et al. (43) compared the Rotazyme I EIA and theRotalex LA test (Finland) with EM and found the sensitivityto be 96% for both and the specificity to be 89% for theRotazyme EIA and 86% for the Rotalex LA test. Sambourget al. (313) tested 204 samples for rotavirus by four tech-niques: two ETAs, Enzygnost and Rotazyme T; and two LAtests, the Slidex Rota-Kit (Biomerieux) and Rotalex (Med-ical Technology Corp.). The positive rates were 47, 38, 37,and 34%, respectively. However, 12 specimens positive bythe Enzygnost test only and 3 specimens positive by theRotalex test only could not be confirmed positive by EM.Brandt et al. (27) found the Slidex Rota-Kit to be 82%sensitive and 100% specific.

Miotti et al. (256) compared 122 samples by three com-mercial tests and their own reference microplate EIA. Thethree tests were the Rotazyme I EIA, the Bio-EnzaBeadEIA, and the Rotalex LA test. The sensitivity was deter-mined to a great extent by the time after the onset of illnessduring which the specimens were collected. There was nosignificant difference in the three tests when they were runon specimens collected early in the patients' illnesses. How-ever, lower degrees of sensitivity were seen with the Ro-tazyme and Rotalex on specimens obtained later in thepatients' illnesses. The lower sensitivity of the Abbott Ro-tazyme was not statistically significant, although the loweredsensitivity of the Rotalex LA was statistically significant. Inaddition, these authors found that a 104 50% tissue cultureinfective dose per 0.1 ml of virus suspension was detected ata 1:300 dilution by Bio-EnzaBead, at a 1:30 dilution by theAbbott Rotazyme, and at a 1:10 dilution by the Rotalex LA.There were no false-positive results with any of the threecommercial tests, and this was seen with newborn as well asother specimens.

Pai et al. (280) compared Rotalex (Finland) with RotazymeI and EM with 165 stools from children and neonates.Rotalex had a sensitivity of 82% and a specificity of 96%compared with EM and was slightly more sensitive andspecific than Rotazyme T. These authors also found that thesensitivity with Rotalex was dependent on the time ofcollection of stool samples relative to onset of symptoms.Sensitivities of Rotalex were 100, 96, 60, and 33% during 1 to4, 5 to 7, 8 to 10, and 11 to 18 days, respectively, after onsetof symptoms, and similar results were observed with Ro-tazyme T. Of 214 EM-negative specimens from asympto-matic newborns, the false-positive rates were 3.3% (7 of 214)for Rotalex and 4.2% (9 of 214) for Rotazyme I.

Several workers have primarily evaluated the Rotalex LAtest. Hammond et al. (145) compared Rotalex (Finland) withEM and found a sensitivity of 92% and a specificity of 98%.However, 19 of 218 specimens could not be evaluated since10 of them gave equivocal results and 9 -of them causedagglutination of the control latex.Julkunen et al. (178), using 570 specimens stored frozen at

-20'C, compared Rotalex LA and a noncommercial EIAused in their laboratory with EM results obtained from these

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samples prior to freezing. They found that their EIA wasmore sensitive than EM (168 versus 145 positive). Of 570specimens, 30 were EM negative and EIA positive, andthese specimens were positive in a confirmatory EIA. Six-teen (2.8%) of the LA-positive specimens were negative byboth EM and EIA, and 15 of 16 were only slightly positive.They concluded that the LA test was good for screening, anddefinitely positive results were reliable.Moosai et al. (258) compared the Rotalex LA test with

EM, their own EIA, and PAGE of viral RNA on specimensfrozen at -70TC. Although they found LA the least complexto perform, it lacked sensitivity and specificity. They sug-gested four modifications to improve the test, includingdiluting the specimens 1:100 rather than 1:10 and reading at20 min, not at 2 min.

Shinozaki et al. (330) compared Rotalex LA with fourother methods: PAGE, EM, SPIEM, and a commercialreverse passive hemagglutination test. The positive rates forthe five methods were 61% (LA), 63% (PAGE), 59% (EM),59% (SPIEM), and 57% (reverse passive hemagglutination).

DeSilva et al. (79) studied Rotalex (Finland) in 90 childrenwith diarrhea and found 90% (80 of 89) agreement with the"established" method(s) of EM alone or in conjunction withthe Enzygnost EIA. Ten percent (9 of 89) were consideredfalse-negative by Rotalex since they were positive by EMalone or by EM with EIA that could be blocked.

Various problems have been associated with ETAs.Yolken and Stopa (414) initially reported problems withnonspecific reactions in ETAs. The nonspecific activity wasmarkedly reduced by pretreatment of the specimens withreducing agents, normal goat serum, and anti-human IgM.The authors concluded that it was likely that the specimenscontained an IgM antibody capable of reacting nonspecifi-cally with other components of the assay. Although pretreat-ment with the mild reducing agent N-acetylcysteine mark-edly reduced this nonspecific activity, such treatment did notreduce the specific EIA activity due to rotavirus.

Studies by Hovi et al. (167) suggested that false-negativeresults might result from fecal protease activity. This prob-lem could be alleviated by adding 1 to 5% bovine serum todilution buffers or by using a synthetic broad-spectrumserine-type protease inhibitor.Hogg and Davidson (162) evaluated false-positive results.

They found that, when they incorporated preimmunizationserum-coated wells as control wells in their EIA, 9.7% ofspecimens giving positive results were eliminated as false-positives.

Several investigators have reported problems with false-positive EIA results on specimens from neonates. Theseoccurred especially in earlier studies with Rotazyme I, inwhich lower positive cutoff values were used. When thecutoff value was raised, some of the problems appeared to beeliminated.Krause et al. (208) reported that 22% (79 of 358) of

neonatal stool specimens from both asymptomatic and diar-rheal neonatal patients were positive by Rotazyme I, al-though of 61 of 79 positives that were analyzed by confir-matory tests, only 7% (4 of 61) were confirmed as positive.This was compared with 77% that were confirmed Ro-tazyme-positive specimens from children and adults. Sug-gested causes of these false-positive Rotazyme tests inneonates included nonspecific binding of rotavirus antibodyto bacteria or staphylococcal protein A in neonatal stools.However, Pai and Mayock (279), using Rotazyme I to

study specimens from infants under the age of 4 months,found that only 9.8% (21 of 214) that were negative by EM

were positive by EIA and thus presumed to be false-positives. However, only 4.5% (9 of 202) were positive whenvisual readings of >1+ were considered positive, as recom-mended by the manufacturer. Similar results were seen byRand et al. (294), who studied stool specimens from diar-rhea-free infants in a neonatal intensive care unit withRotazyme T. None had rotavirus by EM. By EIA, only 6.8%(10 of 147) were considered either low-level positive orsuspect positive. Excluding the suspect positives, whichwere negative on repeat testing, the false-positive rate was4.1% (6 of 147). With five repeatedly positive specimens withsufficient quantity to retest, heating to 560C for 30 mineliminated binding to the Rotazyme bead; heating had noeffect on the Rotazyme-positive control. One highly false-positive result was not changed by heat or other treatment.Thus, the investigators concluded that heat treatment ofpositive samples from neonates could eliminate most of thefalse-positives, although false-positives may result frommore than one cause.

Chrystie et al. (58) found a 15% (8 of 53) false-positive rateon specimens from 5-day-old babies, using Rotazyme T.However, they used an initial cutoff value specified in earlyRotazyme directions; had a revised, higher cutoff value beenused, only 1.9% (1 of 53) of the neonatal samples would havebeen falsely positive (295, 365). In addition, weakly positiveand borderline Rotazyme reactions correlated poorly withdirect EM findings (12, 50, 295).

Rotbart et al. (309) obtained rectal swabs from sympto-matic and asymptomatic babies in a neonatal intensive careunit in which an outbreak of necrotizing enterocolitis andhemorrhagic gastroenteritis occurred. A total of 4.0% (19 of475) of specimens were positive by Rotazyme T, 2.1% (10 of475) from symptomatic babies and 1.9% (9 of 475) fromasymptomatic babies. Confirmatory tests were positive in80% (8 of 10) of the specimens from symptomatic babies,while confirmatory tests were positive in only 33% (3 of 9) ofthe specimens from asymptomatic babies. Differences inRotbart's results and those of Krause et al. (208) may havebeen due to Rotbart's use of swabs, which may havecontained less inhibitory substances, or inhibitory sub-stances may have been less stable on swabs than in the stoolsamples used by Krause et al., or both. Since all of thestudies were carried out at different locales and times,year-to-year and lot-to-lot variations in key reagents mightalso have accounted for some differences. Rotbart et al.recommended that the then current Rotazyme I test not beused for screening asymptomatic infants. They also sug-gested the inclusion of some type of confirmatory testing inthe Rotazyme kit, e.g., reaction with nonimmune serum oruse of monoclonal antibodies.Rudd and Carrington (311), in screening babies in a

neonatal intensive care unit, found that 2.9% (5 of 170) ofbabies had specimens positive by the Rotazyme I test. Twoof these babies had necrotizing enterocolitis, one had bloodydiarrhea, and two were asymptomatic. Thus, a high false-positive rate did not occur in this study.

Giaquinto et al. (126) prospectively studied 500 fecalspecimens from neonates in an obstetrical ward. Superna-tant fluids after 3,000-rpm centrifugation of specimens wereused in the Enzygnost EIA, and 5% (25 of 500) were

positive. Of these, 52% (13 of 25) were confirmed positive bya blocking EIA. All (100%) positive specimens from babieswith diarrhea were also positive in the confirmatory blockingtest, whereas only 33% (6 of 18) from asymptomatic patientswere positive in blocking tests. The authors concluded thatthe Enzygnost test is suitable only in neonates with symp-



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toms, and they did not recommend it for screening asymp-tomatic neonates. They suggested that the Enzygnost andRotazyme test kits include a confirmatory reaction in theirtest kits.Herrmann et al. (151) compared both Rotazyme and their

own EIA, which used a monoclonal detector antibody, withEM. They evaluated specimens from three types of patients:neonates, children, and adults. The sensitivity of their mono-clonal EIA was 100% for all, while that of Rotazyme was100, 86.2, and 50.0% in the three types of patients, respec-tively. The specificity of the monoclonal EIA was 100, 95.6,and 96.5%, while that of the Rotazyme was 37, 95.6, and89.7% respectively. Thus, they concluded that, especiallyfor specimens from children and adults, greater sensitivityand specificity could be achieved with monoclonal antibodyas detector antibody as compared with Rotazyme. Also, useof the monoclonal antibodies eliminated the false-positivereactions seen in specimens from neonates when Rotazymewas used. Herrmann et al. thought that, by using monoclonalantibodies as detector antibodies, the test sensitivity may bedecreased, since these antibodies react with only one epi-tope of a given antigen. This problem could be diminished byusing monoclonal antibody as a capture antibody and apolyclonal serum as the detector antibody, as these investi-gators had reported for a rotavirus RIA. The advantage ofusing the monoclonal antibody for capture is that it may pickup a significant amount of an antigen with several differentepitopes. These investigators considered that the polyclonalsera may contain a number of cross-reacting componentsand may be the cause of false-positives in neonates, sincefalse-positives occur in neonatal specimens in other rota-virus ETAs besides Rotazyme. The disadvantage of usingpolyclonal antibodies as a detector is that they may reactnonspecifically with antigen adsorbed to the solid phase.This may occur even if inhibitors such as serum, serumfractions, or gelatin are included in the diluents to blocknonspecific reactions. For example, they found that in stoolscontaining high titers of rotavirus there was sufficient reac-tivity to give a positive test with microtiter plates coatedwith preimmune sera, even though there was two to threetimes greater reactivity with plates coated with immune sera.None of the studies to date has found false-negative Ro-tazyme results in neonates.Other detection methods. Other testing procedures were

also developed to detect rotaviruses or rotaviral antigens.Some of the earlier tests included counterimmunoelectro-phoresis (237, 250, 251, 339, 366), CF (420, 422), variousimmunofluorescence tests (39, 358, 415), and RIAs (181,249). Counterimmunoelectrophoresis and the CF test arerarely used now, since they are relatively insensitive. TheRIA is sensitive (14, 319) and suitable for the study of largenumbers of specimens, but the need for radioactive reagentsand expensive radiation-counting equipment makes it im-practical in many situations.

Other techniques for detecting rotaviruses in stool sam-ples have been developed. They are primarily researchtechniques and are not used routinely. Among the in vitrotests are an immune adherence hemagglutination test (262), areverse passive hemagglutination test (314, 315), and asolid-phase aggregation of coupled erythrocytes technique(26, 388), and PAGE (258). Various methods to detectrotavirus or rotavii-al proteins in infected cell cultures havebeen developed and include a collodial gold-protein A-IgGtechnique (289), a radioimmunofocus assay (220), and animmunoperoxidase procedure (44). More recently, severalsensitive and specific research techniques for the detection

of rotavirus in clinical specimens have been developed,which include a dot-immunobinding assay (299), a dot-avidin-biotin-amplified immunobinding assay (299), an avi-din-biotin RIA (407), and several dot hybridization tech-niques (82, 100, 116, 217).Antibody detection. Various methods have been used to

detect antibodies to rotaviruses. These have included 1EM(186), the indirect fluorescent-antibody test (23), CF (185),counterimmunoelectrophoresis (62), hemagglutination inhi-bition (HAI) (239), immune adherence hemagglutination(243), reverse passive hemagglutination inhibition (316),neutralization (359), and EIA (417). Neutralization testshave been used extensively for serotyping rotaviral isolatesand are still the standard, although typing now can be doneby EIA.

Several types of tests have been developed to detectrotavirus IgG and IgM antibodies in human sera as well as todetect secretary IgA (sIgA) in sera and stools. These can beused for several purposes: (i) to evaluate the use of serologictests for viral diagnosis, (ii) to study the immune response torotavirus infection, (iii) for epidemiologic purposes, and (iv)to evaluate potential rotavirus vaccines.Yolken et al. developed an EIA test to detect rotavirus

IgG and IgM levels in sera (417) and secretary antibody inhuman milk (410). They immobilized rotavirus antigens onmicrotiter wells by preadsorbing guinea pig antirotavirusserum onto the wells, since adsorption of rotavirus antigensdirectly onto wells did not give reliable results.McLean et al. (246) developed an EIA test to measure

IgG, IgM, and IgA in serum and mucosal secretions (colos-trum, milk, and fecal extracts). Grauballe et al. (129) alsodescribed an EIA to detect sIgA in patient sera. For this test,microtiter plates were precoated with rabbit anti-humanrotavirus serum, followed by the addition of purified humanrotavirus as the capture antigen. Rabbit anti-human secre-tory component, conjugated to horseradish peroxidase, wasthe detector antibody.Inouye et al. (173) described an EIA for detecting IgA in

stool samples. Initially they found that direct adsorption ofpurified rotavirus antigen to a solid phase was unsatisfac-tory. However, they achieved good results by disruptingvirions into small subunits, using the chaotropic agentNaSCN or guanidine HCl, prior to their direct adsorption ofantigen onto microtiter plates.Zentner et al. (419) developed an indirect immunoper-

oxidase assay to detect rotavirus IgG antibody in sera. Theyused SA11-infected MA-104 cells on glass slides and anti-human IgG peroxidase conjugate.Heimer and Cubbit (147) described an indirect immuno-

fluorescent-antibody test to detect IgG and IgM antibodies insera. Bovine rotavirus-infected LLC-MK2 cells grown inmicrotiter plates were used as the substrate. Patient serawere then added, followed by anti-human IgG or IgMfluorescein conjugate.These tests are primarily used as research tools. They are

not commercially available at this time, and there is probablyno great demand for them since their primary use is not asdiagnostic tests.

Immunology

A number of studies have been carried out in the field ofrotaviral immunology. This area of study of the immuneresponse to rotavirus infection has been of great interest,since rotavirus infections appear to be repetitive. What rolevarious immunoglobulins and other factors play in the pro-

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TABLE 2. Serum rotavirus immunoglobulin response to infection

Type of antibody Time antibody first detected Length of time antibody Reference(s)persisted

Neutralizing antibody During convalescence Protection for <1 yr 52Neutralizing antibody During convalescence Not given 59, 292IgM In acute phase, elevated Decreased in convalescence 75, 301IgG In convalescence, elevated Detectable at least 6 to 12 mo 155, 159IgA Not given -12 mo 75IgA Within 1st 2 wk .6 mo 155, 159ScIg Within 1 to 2 wk <4 mo 155, 159sIgA 4 to 10 days 4 to 10 days 129

tection against rotavirus infection is important to know,since oral vaccines are being developed that may conferimmunity to rotavirus infection. Most of the antibody studiesdiscussed below were carried out by EIAs, although RIAand indirect fluorescent-antibody procedures were used in afew studies.The temporal antibody response in the sera of patients

with rotavirus infection has been studied by several groupsof investigators (Table 2). Chiba et al. (52) reported thatprotection against rotavirus gastroenteritis seemed to beserotype specific and related to neutralizing antibody levels.The protective effect was of short duration (<1 year), whichthey concluded was a probable explanation for recurrentattacks of gastroenteritis caused by the same serotype.However, Clark et al. (59) studied the neutralizing antibodyresponse to four human rotavirus types in eight infants afterrotavirus infection. Five originally seronegative infants andthree originally seropositive infants showed an increase inneutralizing antibody to two or more serotypes. No consis-tent pattern of response to different serotypes was detected.Similarly, Puerto et al. (292) studied neutralizing antibodyresponses in 36 convalescent children and found that 19seroconverted to two or more serotypes. Conversion to twoor more types occurred in both initially seronegative andseropositive infants. The investigators suggested that hetero-typic responses may be due to the existence of epitopes thatinduce antibodies capable of neutralizing viruses belongingto different serotypes, since this has been seen with mono-clonal antibodies.

Riepenhoff-Talty et al. (301) and Davidson et al. (75) foundthat, in sera, rotavirus-specific IgM first appeared during theacute phase of infection. This was replaced in convalescenceby rotavirus-specific IgG, while IgM decreased. Convales-cent serum IgG titers were significantly lower in severely illinfants as compared with moderately ill babies (301). Mostchildren still had detectable serum IgG titers after 12months. Hjelt et al. (155, 159) also found that rotavirus-specific IgG was elevated as long as 6 months postinfection.They found that the severity of the illness correlated onlywith the increase of IgG in the serum, a result opposite whatwas reported above (301).

Studies on the role of secretary antibody have been ofspecial interest. Two studies by Hjelt et al. (155, 159)determined that IgA increased in serum within the first 2weeks of illness and persisted in the serum for at least 6months postinfection. Similarly, Davidson et al. (75) foundthat serum IgA dropped significantly or was not detectableby 12 months. Hjelt et al. (155, 159) found that the immuno-globulin secretory component (ScIg) increased in serum 1 to2 weeks after disease onset; however, it had disappeared by4 months. Grauballe et al. (129) detected sIgA in patient serafor only 4 to 10 days after rotavirus was detected in stools,and this correlated well with recent infection. These differ-

ences may have been due to differences in the sensitivities ofthe EIA tests used. Hjelt et al. (155, 159) found that theamount of ScIg in the serum after 1 week correlated with theamount found in the duodenal fluid. Rotavirus IgA in fecesstill persisted in most patients at 6 months postinfection,whereas ScIg had disappeared in the feces of all patientsexcept one.The intestinal antibody response has been studied by other

investigators as well (Table 3). In fecal specimens, Riepen-hoff-Talty et al. (301) found rotavirus-specific sIgA in bothacute and convalescent samples. Davidson et al. (75) re-ported that IgA in duodenal secretions was significantlyhigher in convalescent-phase secretions than in acute-phasespecimens, although duodenal IgM levels were the reverse.In patients with severe or prolonged infection, convalescentduodenal sIgA levels were higher than in patients with mildor moderate disease (301).Sonza and Holmes (338) studied fecal antibodies in four

small children after a family outbreak. Although low levelsof fecal IgG, IgM, and IgA were observed in most cases atthe time of onset, the levels started increasing by 1 to 2weeks, peaked by 3 to 5 weeks, and quickly declined so thatnone were detectable after 2 months.

Stals et al. (344) found that maximum excretion of fecalIgA occurred about day 7 of illness, when virus excretion,which peaked at 2 to 5 days, was subsiding. They concludedthat IgA limits the duration of diarrhea and plays a major rolein intestinal resistance to infection. Shinozaki et al. (331)detected fecal IgA at day 9 postinfection in their patients;excretion of rotavirus had subsided by day 8 in thesepatients. Fecal IgA reached a maximum titer by 2 to 6 weeksand then declined. Yamaguchi et al. (406) found that inprimary infection fecal IgA appeared at 1 week after theonset of illness, and titers reached a plateau between 3 and 5weeks and then declined gradually. In reinfection, IgA titersincreased more rapidly and were maintained at higher levelsfor a longer duration, typical of an anamnestic response. Todetermine whether IgA appearing after primary infectionlimits the infection and plays a role in resistance to reinfec-tion, challenge studies would probably have to be carried outin volunteers or prospective studies would have to be done.Thus, in a prospective study, Hjelt et al. (158) determined

preexisting concentrations of serum rotavirus-specific IgAand IgG prior to the rotavirus "season." They found that,although preexisting IgA did not protect from infection,children with preexisting IgA developed milder cases ofgastroenteritis. Concentrations of IgA measured by EIAwere similar in symptomatic and asymptomatic groups.Rotavirus IgG did not have any protective effect. However,age had a protective effect in that older children had milderdisease.There have been several studies on the levels of rotaviral

immunoglobulins in the serum, milk, and colostrum of



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TABLE 3. Intestinal rotavirus immunoglobulin response to infection

Location of Type of Time antibody first Length of time antibody Reference(s)antibody antibody detected persisted

Duodenal fluid ScIg Within 1 wk; level similar Not given 155, 159to that in serum

Feces IgA Not given .6 mo 155, 159Feces ScIg Not given <6 mo 155, 159Feces IgA Acute phase Throughout convalescence 301Duodenal secretions IgA Acute phase, low Throughout convalescence, higher 75

than in acute phaseDuodenal secretions IgM Acute phase, high Throughout convalescence, lower 75

than in acute phaseFeces IgA, IgM, IgG Low levels at onset; Peaked at 3 to 5 wk; lasted <2 mo 338

increased by 1 to 2 wkFeces IgA Maximum level by 7 days Not given 344Feces IgA Detected by 9 days Peaked at 2 to 6 wk; then declined 331Feces IgA Primary response: de- Peaked at 3 to 5 wk; then gradual 406

tected by 7 days declineAnamnestic response: Peak levels lasted longer

detected sooner

normal mothers and in cord blood and stools of their were low. In bottle-fed infants, no specific IgA was found innewborn infants (Table 4). These studies have been impor- fecal supernatant fluids.tant because of the possible role of immunoglobulins in the Hjelt et al. (157) found that the levels of IgA and ScIg inprotection of infants from rotavirus infection. milk declined from 3 to 4 days postpartum to 2 weeks laterMcLean and Holmes (245) studied 92 mother-infant pairs. and then remained unchanged for 2 months. In duodenal

Rotavirus-specific IgG, but not IgM, was detected in all fluid, rotavirus IgA and ScIg were seen only in a minority ofmaternal and cord sera tested, and this was the only immu- infants at 3 to 4 days and 2 weeks after birth. However, 80%noglobulin that crossed the placenta. Rotavirus-specific IgA of fecal samples contained rotavirus-specific IgA and ScIg,was found in 100% of maternal sera tested, while specific although they disappeared after lactation ceased. Interest-ScIg was found in 53% of the maternal sera. In another ingly, IgA and ScIg can survive proteolysis in the gut. Thus,study, Hjelt et al. (157) found that all of the mothers tested frequent breast meals have a possible protective effect.had IgG and IgA in their sera and 78% had low levels of ScIg Yolken et al. (412) studied secretary antibody (ScIg) toin their sera 3 to 4 days postpartum. Infant serum IgG levels rotavirus in human colostrum and milk. They found rota-correlated well with those of their mothers, but they had no virus-specific ScIg in all colostral samples and most of therotavirus IgA or ScIg in their sera. milk samples. Peak ScIg levels in colostrum fell to low butThese same two groups of investigators also evaluated the detectable levels in milk 1 to 2 weeks postpartum and

protective role of immunoglobulins in the colostrum and remained at these levels for 2 years after delivery, similar tomilk in the same two groups of normal mothers. Rotavirus- results reported above. There were no significant differencesspecific IgG and IgM were found in the colostrum and milk in samples from different geographical areas of the world.of many of the mothers by McLean and Holmes (245), and Cukor et al. (71) studied rotavirus-specific sIgA in moth-these levels dropped off significantly by 3 to 5 days postpar- ers' milk. They found that 80% of the mothers were positivetum. Rotavirus-specific IgA and ScIg were found in all for IgA <1 week after parturition, 50% were positive 7 to 14colostrum and milk samples, except for two mothers who days postpartum, and 24% were positive at 1 month post-were IgA deficient. Specific IgA and ScIg levels in colos- partum. However, as late as 6 to 9 months postpartum, 56%trum-milk dropped off rapidly immediately postpartum to a of those tested were positive. This was due to mothers whosteady low level at 3 to 4 days postpartum. In breast-fed were initially negative eventually becoming antibody posi-infants, specific IgA was found in fecal supernatant fluids tive, possibly because of a subclinical rotaviral infection.after 2 days, even when maternal milk IgA concentrations Thus, two routes of passively transferred rotavirus antibody

TABLE 4. Rotavirus immunoglobulins in normal mothers and newborns

Location of Type of Time antibody first Length of time antibody Referenceantibody antibody detected persisted

Colostrum/milk IgG, IgM Not given Dropped off by 3 to 5 days postpartum 245Colostrum/milk IgA, ScIg Not given Dropped off by 3 to 4 days postpartum to 245

steady low levelFeces of breast-fed infants IgA At 2 days Not given 245Colostrum/milk IgA, ScIg Not given Declined from 3 to 4 days postpartum to 2 157

wk later; then remained unchangedInfant duodenal fluid IgA, ScIg At 3 to 4 days At least 2 wk 157Colostrum/milk ScIg Not given Fell to low but detectable levels 1 to 2 wk 412

postpartum and remained unchangedfor 2 wk

Colostrum/milk IgA Not given Continued dropping off for 7 to 30 days 71

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were identified by these studies, serum and colostrum-milk.However, serum antibodies have doubtful protective capac-ity (118).

In addition to specific antibodies, nonspecific factors inmilk and in the gut may play a role in the protection ofinfants against rotavirus infection. Studies by McLean andHolmes (247) suggested that both sIgA and trypsin inhibitorsin human milk may protect neonates against rotavirus infec-tion during the first 5 days of life. They found that breast-fedbabies were less likely to become infected with rotavirus andshowed lower stool tryptic activity or higher antirotaviralsIgA antibody, or both, than bottle-fed babies.While the above studies concentrated on the levels of

various rotavirus-specific immunoglobulins in body fluids,these levels were not correlated with their possible role inthe protection against, or modification of, rotavirus infectionand disease.

Thus, several studies have been done on the role ofspecific rotaviral antibody in rotavirus infection. Chrystie etal. (57) studied rotavirus infection in an endemic situation ina neonatal nursery. They found that breast-fed babies ex-creted rotavirus significantly less frequently than those whowere formula fed. Also, if infection did occur among breast-fed infants, they excreted less virus than infected formula-fed infants. More specifically, in a second publication, thisgroup (363) reported on the role of specific antibody in cordblood and breast milk in this same outbreak of rotavirusgastroenteritis. The ranges, distributions, and geometricmean titers of serotype 2-specific IgG in cord blood andserotype 2-specific IgG and IgA (97% positive) in the moth-ers' breast milk were similar among rotavirus-positive androtavirus-negative neonates. Interestingly, there was nocorrelation between the amount of milk IgA antibody and theamount of neonatal viral excretion. These studies suggestthat factors other than rotavirus antibody in breast milk areof importance in preventing rotavirus infection in neonates.To determine which nonspecific factors may play a role in

resistance to rotavirus infection, these same workers (364)evaluated the role of neutralizing activity, nonimmunoglob-ulin antiviral activity, and a1-antitrypsin activity in themothers' milk. They were unable to correlate any of thesefactors with protection from rotavirus infection. However,they found that rotavirus in infected stools from breast-fedinfants was frequently clumped, although that in stools fromformula-fed infants was rarely clumped (361). Thus, whenthey specifically studied human lacteal rotavirus antibodiesby IEM (361), they found that the IgA- and IgG-containingfractions in human and bovine milk samples caused aggre-gation of rotavirus particles. Clumping did not occur withantibody-negative milk or with the nonimmunoglobulin frac-tions of antibody-positive milk. They suggested that adsorp-tion to susceptible cells of viral aggregates formed in thepresence of low levels of antibody and containing unneutra-lized virus was one mechanism by which infection in thepresence of antibody might occur.

Similar results were found by Weinberg et al. (392). In 50infants who had rotavirus gastroenteritis within the first yearof life, 64% had been breast-fed and 70% had not. Therewere no significant differences between the two groups in theaverage age of infection, the mean duration of diarrhea, themean number of bowel movements in 24 h, or the frequencyof fever or irritability. The only apparent difference betweenthe groups was that the frequency of vomiting was signifi-cantly decreased in the breast-fed children. Their resultssuggested that breast-feeding offered little protection againstrotavirus gastroenteritis. Similarly, Duffy et al. (90) found

that, although rotavirus attack rates were similar betweenbreast- and bottle-fed infants, rotavirus infection in breast-fed infants was milder and of shorter duration than inbottle-fed babies.

In addition to studies on the role of passively acquiredmaternal antibody in neonatal infection, studies on the roleof actively acquired antibody in preventing or modifyingrecurrent infection have been carried out. Bishop et al. (17)studied the ability of neonatal rotavirus infection to conferimmunity to postneonatal rotavirus infection. Fifty-five per-cent of infants with neonatal infection and 54% of thosewithout infection developed rotavirus infection during thefollowing 3 years. Babies with reinfection had symptomsthat were significantly less severe than those who had noprevious neonatal infection. They concluded that neonatalrotavirus infection does not confer immunity against reinfec-tion, but does protect against the development of clinicallysevere disease during reinfection.

In addition to the study of natural passive immunity,studies on the use of artificial passive immunity for preven-tion and treatment of rotavirus infection have been carriedout. Barnes et al. (8) evaluated the use of human gammaglobulin or placebo given in each feed during the first weekof life to 75 low-birth-weight infants in a nursery whererotavirus was endemic. Twenty-five of the babies excretedrotavirus during the first 2 weeks of life and were consideredthe "challenge" group. Gamma globulin administration wasassociated with delayed excretion of rotavirus and withmilder symptoms. The diarrhea necessitated low-lactosefeeds in 55% (6 of 11) of placebo babies but in only 7% (1 of14) gamma globulin-treated babies. The authors concludedthat oral human gamma globulin seemed to protect low-birth-weight infants from rotaviral diarrhea.

In another study (223) two children and one adolescentwith primary immunodeficiency syndromes and prolongedexcretion of rotavirus were given a single oral dose of humanserum immunoglobulins. The result was the generation ofrotavirus-specific immune complexes in the stools with asubsequent decrease in the presence of uncomplexed rota-virus antigen. The clinical efficacy of this treatment was notdetermined. However, the authors concluded that the oraladministration of immunoglobulins with specific reactivitieshad potential for prevention or treatment of gastrointestinalinfections.

Ebina et al. (96, 97) immunized pregnant cows with humanrotavirus to prepare rotavirus-specific IgA-rich cow colos-trum. In a therapeutic trial, the rotavirus-specific cow colos-trum had no effect on the duration of diarrhea, bowelmovements, or virus shedding. However, when used pro-phylactically, good results were obtained. Only 17% (one ofsix) of infants developed rotavirus diarrhea, whereas 86%(six of seven) of controls given market milk as a placebodeveloped diarrhea.

Similarly, Brussow et al. (38) immunized pregnant cowswith four human rotavirus serotypes. They then preparedfreeze-dried lactation milk in which rotavirus-neutralizingactivity was 10 times higher than that of pooled humanimmunoglobulins. When the milk concentrate was used totreat infants with rotavirus gastroenteritis (154), there was areduction in the duration of virus excretion and of diarrhea.Yolken et al. (411) found detectable levels of rotavirus IgG

in both raw and pasteurized milk, but little or no antibody incommercial infant formulas. In vitro, the milk samplesinhibited the replication of human and bovine rotaviruses intissue culture. In vivo, the milk samples decreased or



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prevented diarrhea in infant mice infected with virus-milkmixtures.

Offit and Clark (274) found that, when pregnant mice wereinoculated with rotavirus and their newborn mice werechallenged, the maternal antibody protection was dependenton both antibody titers and the serotype, with protectionbeing primarily to the homologous strain. Similarly, Lo-sonsky et al. (225) found that incubating murine rotaviruswith homologous mouse antisera prior to infection preventedsymptomatic disease, whereas heterologous antisera did notneutralize the virus as effectively. Asymptomatic mice,however, developed an immune response.

In contrast to the number of studies on the role ofantibodies in rotavirus infection, there are few reports on thecellular immune response. Totterdell et al. (362) studied thelymphoproliferative responses to rotavirus antigen in wholeblood taken from several types of individuals. These in-cluded healthy adults, elderly patients at geriatric institu-tions, adult renal transplant patients, one child with severecellular immunodeficiency who had been excreting rota-virus, and cord blood from healthy babies. Using a lympho-cyte transformation assay, they found that elderly andtransplant patients had significantly lower lymphoprolifera-tive responses to rotavirus antigen as compared with healthyadults. However, after acute rotavirus infection, the oneinfected transplant patient and the elderly patients had good,though transient, lymphoproliferative responses and goodspecific antibody response. The cord blood samples showedno lymphoproliferative responses to rotavirus, although theycontained specific antibodies. The immunodeficient childhad neither an immunoproliferative nor an antibody re-sponse to rotavirus antigen.

Results similar to those seen in the healthy adults in theabove study were seen in newborn mice infected withrotavirus in another study (303). A virus-specific cell-medi-ated response appeared in splenic lymphocytes 2 days afterinfection and peaked at 10 days postinfection. This peak ofactivity closely followed the cessation of diarrhea, suggest-ing that cellular immunity may function as a limiting factor indisease (303). However, Eiden et al. (99) found that neonatalT-cell-deficient (athymic) mice developed a self-limited ro-tavirus infection identical to that in immunocompetentmouse controls (99). Their athymic mice developed noserum or intestinal antibody, whereas the normal controlsdid. They suggested that recovery from rotavirus infection inmice is not dependent on functional T cells or on specificantibody, but rather is mediated by nonimmune mechanismsor macrophages. Thus, the mechanisms involved in recoveryand protection from rotavirus infection are complex, involv-ing an interplay between local and systemic and humoral andcell-mediated responses, and also nonspecific factors (362).

Prevention

Vaccines. Since rotavirus is a serious problem in develop-ing nations and is the single most important etiologic agent ofacute gastroenteritis requiring hospitalization in developedcountries (188), there is a need to control this disease by useof a rotavirus vaccine. Vaccines would be administeredorally, since this is the natural route of infection and thisroute would stimulate local intestinal IgA antibody (188).

Several groups have been developing experimental oralvaccines for human use. In Europe, a collaborative group ofFinnish and Belgian investigators (376-379, 382) have beenevaluating an attenuated bovine rotavirus vaccine strain,RIT 4237. The rationale for a heterologous rotavirus vaccine

has been (i) the ease with which animal rotaviruses can bepropagated in vitro and (ii) their sharing of a common groupantigen with most human rotavirus strains (group A) (378).This group antigen is the major inner capsid protein VP6,which is also the distinct subgroup antigen. RIT 4237 is asubgroup I rotavirus.The RIT 4237 vaccine strain was passed 147 times in

primary bovine kidney cells, was then passed 7 times inprimary Cercopithecus monkey kidney cells, and was usedat pass 154 (362) with a mean titer of 1081 50% tissue cultureinfective doses per oral dose. After evaluation of the safetyof the vaccine in adults, 17 seronegative 2-year-old childrenwere given a single oral dose. In this preliminary study, 70%of the children seroconverted unequivocally and 18% had apossible seroconversion, for a total seroconversion rate of88%. None of the children had a major clinical reaction tothe vaccine; i.e., no gastrointestinal or constitutional symp-toms occurred. None of the children excreted rotavirus,although three individual stools were positive for antigen byEIA.

In a larger group of 178 pediatric patients aged 8 to 11months given the vaccine during the rotavirus season, theprotective effect of the vaccine was evaluated (379). Of theseronegative infants, 47% of the vaccine recipients serocon-verted, but there was an 88% protection rate from clinicallysignificant rotavirus diarrhea. In this study, although itappeared that serum antibody could be used as a marker ofsuccessful vaccination, protection against clinical diarrheacould not be strongly associated with these serum antibod-ies. Vaccination and subsequent naturally acquired immu-nity protected against clinical diarrhea. However, vaccina-tion did not protect against subclinical infection, asdetermined by a booster response or by antibody serocon-version.

In a pre-rotavirus season vaccination study carried out inthe autumn, infants aged 6 to 12 months were given eithertwo doses of the RIT 4237 vaccine or a placebo (377).Results similar to those of the previous study were seen.Vaccinees who failed to seroconvert had less rotavirusdiarrhea than placebo recipients, suggesting that immunitymay be mediated by factors other than serum EIA antibody.The protection rates against clinically significant diarrheacaused by serotypes 1, 2, and 3 were 72, 100, and 100%,respectively.The RIT 4237 vaccine appeared to protect against several

human rotavirus serotypes. In one study, the subgroup I RITvaccine offered protection during an outbreak of subgroup IIrotavirus (379), which is the most prevalent subgroup seen inthe United States. In another study, limited evidence sug-gested that the RIT vaccine offered protection against se-rotypes 1, 2, and 3 (377). Preliminary data also indicated thatthis vaccine may offer protection for at least 2 years (376).

Since the RIT 4237 strain is relatively acid labile and maylose infectivity if exposed to high gastric acidity occurringbetween feedings in infants, the investigators felt that anappropriate acid-neutralizing substance such as milk mightincrease the seroconversion rate. Thus, a RIT 4237 vaccinewith 108 3 50% tissue culture infective doses was given tobreast-fed, formula-fed, and fasting infants (382). A neutral-izing antibody response occurred in 69% of the breast-fed,100% of the formula-fed, and 63% of the fasting infants, withan overall response rate of 77%. The breast milk wassomewhat inhibitory, since it may give partial protectionagainst naturally occurring rotavirus diarrhea. Poorer re-sponses occurred when the vaccine was used containing107-2 or 106.3 50% tissue culture infective doses per dose.

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Basically, the European bovine RIT 4237 vaccine was safe,but not as immunogenic as desired, lacking immunogenicityin some infants and children. This vaccine has not beenmarketed commercially.Another candidate rotavirus vaccine has been developed

and evaluated at the National Institutes of Health (5, 55, 184,224). The vaccine strain is also an animal virus, rhesusrotavirus-1 strain MMU-18006. The virus was derived fromthe stool of a 3-month-old rhesus monkey with diarrhea. Itwas passed nine times in primary or secondary monkeykidney cell cultures and seven times in a semicontinuousfetal rhesus monkey diploid lung cell strain, DBS-FRhL-2,developed by the Division of Biologic Standards (184). Themajor neutralizing protein is closely related or identical toVP7, the major outer capsid protein detected by cross-neutralization (380) of human serotype 3 (184, 224). Thisvaccine is more immunogenic than the RIT 4237 vaccine, butit also produces more side reactions.

In initial studies, the MMU-18006 strain was found to beattenuated in newborn and juvenile rhesus monkeys. It wasfirst evaluated in human young adults and then children aged2 to 12 years and was found to be immunogenic in >80%, butcaused no side reactions (224). The vaccine, containing 105PFU per dose, was then tested in 14 infants aged 5 to 20months; 13 infants were given placebo. It was administeredafter the infants received formula buffered with sodiumbicarbonate. There was a fourfold or greater rise in serumantibody titer in all of the vaccinees, but none in the placebocontrols. The primary side reaction to the vaccine was thedevelopment of fever above 100.60F (37.80C) 3 to 4 dayspostvaccination. All vaccinated children excreted rotavirusin their stools within 1 week of vaccination.

In a similar study of 13 vaccinated infants and 10 controls,significant rises in serum antibody titers occurred in allvaccine recipients, although they also exhibited side effectsto the vaccine (5). These included rhinorrhea, passage of alarger than usual number of stools, and passage of moresemiformed or unformed stools as compared with controls.All vaccinees shed rotavirus in their stools.The MMU-18006 vaccine appeared to be more immuno-

genic but caused more side reactions in American popula-tions than the RIT 4237 vaccine did in Finnish recipients.One difference between the two populations was that, in theFinnish studies, most of the vaccine recipients were initiallyseronegative as determined by EIA, and many of the remain-ing vaccinees had only low levels of detectable antibody,whereas higher percentages of the American study grouppopulations had detectable rotavirus antibody prior to vac-cination. Since the vaccine populations differed, a collabo-rative study was carried out comparing the two vaccines inone study population in Finland (380). Results of bothvaccines were similar, with the RIT vaccine being lessimmunogenic but showing fewer side reactions, while theMMU vaccine was more immunogenic with more adverseside effects.Adverse reaction rates to the MMU-18006 vaccine were

lower, however, in a Venezuelan study. This was dueperhaps to differences in preexisting immunity due to mater-nally transferred antibody in the vaccinees in that country.Thus, the MMU-18006 vaccine might be suitable for thistype of population (380). The MMU vaccine might also beuseful in developing countries, when the only chance tovaccinate might be simultaneously with the oral polio vac-cine, which might interfere to some extent with the rotavirusvaccine. The MMU vaccine might also be effective in earlyinfancy, when passively acquired antibodies are at a high

level and when less side reactions may occur. In such apopulation, a more attenuated vaccine may not take, as wasshown to be a problem with the RIT 4237 vaccine (380).However, the RIT 4237 vaccine did seem to offer protectionagainst infection in infants in highly developed countrieswhen the infants had little or no preexisting antibody. Thehigh level of protection seen in spite of the lack of serumantibody response may be due to protection caused byfactors such as local IgA antibody and cell-mediated immu-nity.

Other rotavirus vaccines are also being considered. Clarket al. (60) reported on a vaccine developed from bovinerotavirus strain WC3, adapted to grow in cultures of CV-1cells, a line of African green monkey kidney cells. Thevaccine, used after the 12th cell culture passage, contained 3x 107 PFU per dose and was initially tested in adults andolder children. After it was tested in 52 infants and childrenaged 5 months to 6 years, no clinical sequelae occurred, andviral shedding was found in only 30% of the vaccinees.Serum neutralizing antibody was induced to the WC3 strainin 95% of the infants aged 5 to 11 months and to humanserotype 3 virus in 50% of the vaccinees. Preexisting anti-body to human serotype 1 or 3 frequently exhibited a boosterresponse.The use of reassortant vaccines has also been proposed by

the National Institutes of Health group (254, 255), sincerotaviruses have a segmented genome which can undergogenetic reassortment in vitro (188). This group coinfectedcell cultures with (i) the cultivatable bovine rotavirus strainUK and (ii) one of several "noncultivatable" human rota-virus strains representing serotypes 1, 2, and 3. Monoclonalantibodies to the major outer capsid neutralization glycopro-tein VP7 were used to select reassortants with humanrotavirus-neutralizing specificity. The technique yieldedmany reassortants which received only the gene segmentcoding for the major neutralization protein for the humanparent strain and the remaining genes for the animal rota-virus parent strain. Reassortants of this type representpotential vaccine strains. Theoretically, additional reassor-tant rotavirus vaccines containing VP4 and VP7 from twoantigenically distinct rotavirus parents might also protectagainst disease induced by two or more serotypes (276).

Epitope-specific immune responses to rotavirus vaccinehave been studied by Shaw et al. (326). They used serotype-specific monoclonal antibodies directed at VP7 in a compet-itive-binding EIA to measure epitope-specific immune re-sponses to serotypes 1, 2, and 3 in sera of children whoreceived a serotype 3 vaccine. Antibodies to serotype 3 weredetected in 72% of the sera, and antibodies to serotypes 1and 2 were detected in only 11% of the sera. Also, specificmonoclonal antibody to VP4 (originally called VP3), whichneutralizes three serotypically distinct strains of rotavirus,was used to detect the presence of similar antibodies in 56%of the test sera. This finding suggests a mechanism ofheterotypic immunity to rotavirus vaccination.

Chemical disinfection. The prevention and control of rota-virus infection by chemical disinfection have been studied bySpringthorpe et al. (342). They studied the effects of 76chemical disinfectants on suspensions of virus in the pres-ence or absence of tryptose phosphate broth (peptides andinorganic salts) or fecal matter. Thirty-two percent of thedisinfectants were considered highly or moderately effectiveand inactivated at least 106 PFU of virus in 1 min. However,the remaining 68% were effective only in the absence oforganic matter or were completely ineffective. The sameinvestigators (221) also evaluated 27 disinfectants for their



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ability to inactivate rotavirus on inanimate objects, usingdisks of stainless steel, glass, and plastic contaminated with107 PFU in fecal matter which was allowed to dry. Only 9 ofthe 27 disinfectants reduced the rotavirus titer by 6 logo,while the others were ineffective. These nine disinfectantsare used for specific purposes in a variety of products forhome, hospital, and food service use. They include hydro-chloric acid, peracetic acid, isopropyl alcohol, chlorhexidinegluconate, glutaraldehyde, chloramine-T, povidone-iodinecomplex, sodium o-benzyl-p-chlorophenate, and a quater-nary ammonium compound. Since rotavirus was resistant toa wide range of commonly used chemical disinfectants, it isimportant to use those chemicals that are effective to preventand control outbreaks of rotavirus disease.

Treatment

Although oral gamma globulin and bovine colostrum havebeen used experimentally in the treatment of rotavirusgastroenteritis as described above, these are not the main-stay of treatment of viral gastroenteritis. Morbidity andmortality in patients with rotaviral and other viral gastroen-teritides are primarily due to dehydration and electrolyteimbalance (90, 206). Thus, the primary purpose of therapy isto correct these problems by providing adequate hydrationto maintain blood volume, electrolyte homeostasis, andacid-base balance (90). Replacement of water and electro-lytes in dehydrated patients can be done with either intrave-nous therapy or oral electrolyte solutions, depending on theclinical situation.A child with diarrhea must first be assessed for his/her

degree of dehydration. This includes evidence of weightloss, changes in skin turgor, sunken eyes, dry mucousmembranes, absence of tears, decreased urine output, andchanges in mental status or vital signs or both (90).

Intravenous fluid therapy for significant dehydration andfasting have been the standard therapy in developed nationsfor over 40 years (90, 218). Following this, a period of fluidmaintenance and a gradual return to a normal diet is advo-cated. The rationale for this approach is to provide bowelrest and to prevent the possibility of food-induced malab-sorption (360). Intravenous therapy is needed for severelysick children, who are (i) in shock and unable to drink fluids,(ii) persistently vomiting, and (iii) having stool losses of>100 ml/kg per h and (iv) who cannot tolerate oral fluids(360, 396).However, the routine use of intravenous hydration for less

severely ill children has been questioned recently. Often oralsugar-electrolyte rehydration solutions are used in theUnited States for maintenance therapy of mild diarrhea withdehydration, as an interim feeding prior to grading to anormal diet, or are incorporated with early refeeding torestore nutritional losses (218, 360, 396).

Oral glucose-electrolyte solutions have been used for 25years in the treatment of dehydration due to acute infantilediarrhea including rotavirus diarrhea. Two basic types oforal electrolyte solutions are now used in the United States.The first type is "rehydration" fluids with a sodium contentof 60 to 90 meq of sodium per liter, and the second is"maintenance" fluids of 30 to 50 meq of sodium per liter.The glucose content in both solutions is 2.0 to 2.5% (gramsper deciliter); higher amounts are undesirable since they can

produce osmotic diarrhea (396). Glucose-coupled sodiumtransport is the physiological basis for oral rehydrationsolutions, since glucose enhances sodium transport in thesmall intestine.

The oral glucose-electrolyte rehydration fluids are effec-tive, inexpensive, and widely available for hospital or homeuse. Other advantages are that they avoid the cost and risk ofintravenous therapy and they stimulate recovery of smallbowel mucosal absorptive function (396).

In one study, Listernick et al. (218) studies 15 patientstreated with oral solution as outpatients in an emergencyroom holding room and 14 matched controls treated asinpatients on intravenous therapy. Rotavirus was detected in11 of 15 orally dehydrated patients and in 10 of 14 intrave-nously rehydrated patients. The group found that 13 of 15 ofthe orally treated patients were successfully rehydrated inthe emergency room in 10.7 h at a cost of $275 versus 103.2h at $2,300 for the inpatients.

In underdeveloped or developing countries where thereare limited medical resources and where malnutrition iscommon, oral rehydration therapy and continued feedinghave been advocated (360). For marginally nourished ormalnourished children, diarrhea associated with starving canhave deleterious effects. When a person has fasted for 3 to 5days, depletion of intestinal digestive enzymes and gut massoccurs, and absorption and digestion of glucose, salt, water,amino acids, and disaccharide is substantially reduced (360).Besides the negative effects of fasting, there are somepositive effects of feeding a child who has diarrhea in thatintraluminal nutrients induce intestinal digestive enzymesand cell proliferation (360). Thus, in underdeveloped coun-tries, continued feeding is an important adjunct to oralrehydration therapy.

Older children with mild to moderate dehydration can betreated at home with oral fluids. Sugar-containing softdrinks, juices, and tea can be used. Since these drinksusually have a low sodium content (0.5 to 20 meq/liter),additional sodium supplement from salted crackers, bouil-lon, or soup is needed. However, in a small infant withsevere diarrhea, more precise fluid and electrolyte replace-ment is necessary (396).

Children with acute diarrhea are often fasted to reduceconsequences of malabsorption, acidosis, fluid loss, anddepleted bowel and mucosal injury. However, advocates ofearly feeding believe that they can prevent or minimize thedeficit of calories and protein, maintain or stimulate therepair of the intestinal mucosa, and maintain the brushborder by avoiding a prolonged fast (396). The AmericanAcademy of Pediatrics recommends that, if the child isstable, refeeding should not be delayed for more than 24 hunless there is significant dehydration, severe vomiting, orabdominal distention (396). Feedings should begin withbreast milk or dilute formula and advance slowly to full-strength formula over 2 or 3 days. Nonlactose carbohydratebland solids such as rice, cereal, and potatoes should bereintroduced as soon as they are tolerated. Bland foods arethought to have a fast emptying time in the stomach and lesslikelihood of stimulating peristalsis. Also, frequent smallfeedings are less likely to distend the stomach and causesubsequent vomiting. Small feedings also present the smallintestine with less lactose load at any one time (396). Infantsrequire close observation for evidence of transient, mild,secondary lactose intolerance when starting feeding or for-mula, especially with rotaviral disease. Evidence of lactoseintolerance includes a marked increase in stools, abdominaldistension, frothy stools, or flatulence. An acidic stool pHand a positive Clinitest (Ames Div., Miles Laboratories,Inc., Elkhart, Ind.) for sugar further suggest lactose intoler-ance (396).

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ADENOVIRUSES

Introduction

Adenoviruses are 70- to 75-nm, nonenveloped, icosahe-dral, ds deoxyribonucleic acid (DNA) viruses classified inthe family Adenoviridae. Adenoviruses infect most speciesof mammals, birds, and amphibians (386). Human and othermammalian adenoviruses are in the genus Mastadenovirus.There are 41 known distinct serotypes of human adenovi-ruses, now considered to be 41 species (386). The firstadenovirus was originally isolated from adenoid tissue, andadenoviruses have been associated primarily with respira-tory disease, as well as with ocular and genitourinary tractinfections. Early reports also associated adenoviruses withdiarrhea in infants and children, but actual causation was notdetermined (177, 257). These adenoviruses associated withdiarrhea could be isolated in routine cell cultures and con-sisted of lower serotypes.During the last 13 years, adenoviruses have been detected

in stools of patients with gastroenteritis (33, 108, 110, 119,197, 232, 297, 300, 324), using the technique of EM (Fig. 1B),but often, they could not be isolated in routine cell cultures.These viruses that are not readily cultivable are calledenteric or fastidious adenoviruses and represent new types,40 and 41 (77). Some investigators have suggested using theterm "fastidious" adenovirus for those adenoviruses thatare detected by EM, in EIAs specific for types 40 and 41, orin special cell cultures such as 293 cells, but which cannot bepropagated in commonly used routine cell culture (228). Thegeneral term "enteric" adenovirus is often used for anyadenovirus observed in stool by EM, in which cell culturepropagation is not attempted. This terminology will befollowed in this review.

Molecular Biology and Classification

Most of the research on the molecular biology of adeno-viruses has been carried out on adenovirus serotypes 1 to 39.However, much of the basic molecular biology of theseserotypes applies to the fastidious types 40 and 41 as well.Adenoviruses, including the fastidious ones, are 70- to 75-nmicosahedral viruses made up of 252 capsomeres. The outercapsid is composed of two main capsomere types. The firsttype is the 240-nonvertex hexon capsomeres and the secondtype is the 12-penton base capsomeres which form the 12vertices of the icosahedran. A fiber vertex projection pro-trudes from the base of each penton (387). The dsDNA corecontains the linear dsDNA with a molecular weight of 20 x106 to 25 x 106 (387).The capsid components have antigenic determinants with

a wide range of specificities. The hexons are responsible forgroup, subgroup, and type specificities (387). Mammalianadenoviruses share a common group-specific determinantthat can be measured by the CF test. The antigens thatspecify the adenovirus serotypes are located on the hexonand are measured in the neutralization test. The pentoncapsomeres carry group and subgroup specificities. Thisgroup specificity is also shared by mammalian (human,simian, and canine) adenoviruses. The fibers carry subgroupand type specificities. This type specificity is measured bythe HAI test (387).There are 41 different adenovirus serotypes (77) consid-

ered to be species by Wadell (386). The 41 types share thegroup antigen, which can be detected by CF tests. Thedifferent serotypes were distinguished initially by neutrali-

zation tests, although RIAs and EIAs have been developedto distinguish types 40 and 41.The adenoviruses were first classified into groups or

subgroups in several ways (168, 308, 387). Huebner (168)divided the human adenoviruses into subgroups based ontheir oncogenicity for newborn hamsters. Subgroups A andB contained "highly" and "weakly" oncogenic adenovi-ruses, respectively. Nononcogenic adenoviruses that trans-formed rodent cells in vitro were classified into two sub-groups, C and D (244). Interestingly, this classificationcorrelates with the subdivision of adenoviruses based on theG+C content of their genomes (130, 290).

Since the above grouping of adenoviruses is based onproperties representing <6% of the genome, Wadell et al.(386, 387), studied the adenovirus relationships based ontheir virion polypeptides. These polypeptides are encodedby a major portion of the adenovirus genome. Based on theirpolypeptide pattern, adenovirus types 1 to 39 fell intosubgroups A to D, with the exception of type 4, whosepattern did not fit and thus was classified into subgroup E. Afastidious adenovirus isolated from diarrheal stools studiedby Wadell also had its own distinctive characteristic poly-peptide pattern and was therefore placed in subgroup F (type40).Green et al. (131) also identified five groups, A to E, based

on the DNA relatedness among 31 adenovirus types. Wadellet al. (386, 387) compared the restriction endonuclease digestpatterns of their subgroup F fastidious adenovirus with othersubgroups. They found that their fastidious adenovirus dis-played a unique restriction pattern with no resemblance toother adenovirus types and thus should be relegated to itsown subgroup.De Jong et al. (77) and Uhnoo et al. (369), using restriction

endonuclease analysis, determined that fastidious adenovi-ruses formed two different species (serotypes) and sub-groups. Serotype 40 was placed in subgroup F and serotype41 was placed in subgroup G. However, these two typeshave biological similarities and are indistinguishable by HAItests (192). Thus, types 40 and 41 appear to have very similarDNA sequences coding for their antigenic determinants eventhough their genomes are apparently different as seen byrestriction endonuclease digestion (194).

Takiff et al. (351) carried out physical mapping of types 40and 41 and found that, although their restriction profileswere different, they appeared to have several cleavage sitesin common. Cross-hybridization studies showed consider-able homology between types 40 and 41, but much lesshomology occurred between these two types and adenovirustype 2. Van Loon et al. (375) found that the DNA homologybetween the two types was 62 to 69% and recommended thatadenovirus type 41, previously classified as subgroup G, beclassified together with type 40 in subgroup F. These sub-groups have also been referred to as subgenera (271, 375).

Epidemiology

Adenoviruses were among the first viral agents associatedwith acute nonbacterial infectious gastroenteritis (177, 257).In the 1960s, Moffet et al. (257) detected adenovirus in 17%of infants <2 years of age with diarrhea compared with 5% ofcontrol infants. However, these adenoviruses were types 1to 31, with lower-numbered types predominating, and allwere cultivated in routine cell cultures. Kapikian et al. (185)detected adenovirus by EM in 11% (16 of 143) of infants andyoung children with diarrhea, although in this early studyculture was not carried out and there was no control group.



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Spratt et al. (341) found adenovirus by EM in 17% (7 of 41)of infants with diarrhea versus 5% (4 of 74) of asymptomaticcontrols. CPE was detected in six of eight inoculated HEp-2cultures, and the viruses isolated were types 2 to 7.

In the late 1970s, several groups reported that someadenoviruses in diarrheal stools of pediatric patients thatwere detected by EM could not be cultivated. Flewett et al.(110) first reported a nosocomial outbreak of gastroenteritisin 32% (6 of 19) of patients in a long-stay children's ward ofan orthopedic hospital in England. Adenovirus was detectedin four of six sick children, but in none of the asymptomaticcontrols. Attempts to culture the virus in a number of cellculture types failed except that subtle CPE was seen in someHEp-2 cultures 10 to 20 days after inoculation. These effectswere not seen on passage. In 3 of 30 patients in Johannes-burg, South Africa, Schoub et al. (324) detected, by EM,adenoviruses that were fastidious. Although they producedCPE with typical nuclear inclusion bodies in human embry-onic fibroblast cultures, they could not be passaged or typed.By EM, Middleton et al. (253), in Toronto, Ontario,

Canada, found adenoviruses in 12.7% (85 of 669) of virus-positive stools from pediatric patients with gastroenteritis.The authors did not claim that all of these adenoviruses weretrue causative agents of disease since no control populationwas studied. However, adenoviruses were seen more fre-quently by EM than could be isolated in cell culture. Theauthors also noted that adenoviruses were sometimes ac-companied by what appeared to be adenovirus satelliteviruses.Madeley et al. (232), in Glasgow, Scotland, detected

adenovirus in 11.5% (21 of 183) of babies under 2 years ofage with diarrhea; 9 adenoviruses were detected only by cellculture, 10 were detected only by EM, and 2 were detectedby both methods. Those detected only by EM showed someevidence of growth in rhesus monkey kidney, human am-nion, and/or human embryonic kidney (HEK) cells by CPE,but they could not be passed or typed; EM of the cellcultures did not suggest viral replication. It appeared tothese investigators that the chance of growing adenoviruswas inversely related to the amount of virus seen by EM.This observation regarding fecal adenoviruses has beenmade by others. Bryden et al. (39) found that many fecalsamples containing adenoviruses that could not be isolatedin cell culture contained large numbers of particles, up to1011 virions per ml.

In a four year study in Washington, D.C., Brandt et al.(33), detected significantly more uncultivatable adenovirusesin pediatric inpatients with gastroenteritis (5.1%, or 31 of604, only 5 of which grew in cell culture) than from inpatientcontrols (1.9% or 10 of 522, 6 of which grew in cell culture).Similar results were seen in outpatients with gastroenteritis.This strongly suggested that these noncultivable virusesplayed a role in acute enteric disease.

Similarly, Richmond et al. (300) reported a 2-month out-break of gastroenteritis in 17 of 24 children under 2 years ofage at a Royal Air Force station in the United Kingdom inwhich poorly cultivatable adenoviruses were detected byEM. None of the adenoviruses could be serially propagatedin baboon kidney, HeLa, human embryonic lung, or HEKcells. However, a transient CPE typical of adenovirus de-veloped in the HEK cultures inoculated with stool samplescontaining large numbers of adenovirus particles, and theaffected cells were stained by indirect immunofluorescencewith specific adenovirus antiserum.

In a retrospective, 4-year study of 2,606 stool specimensfrom patients with acute gastroenteritis in Toronto, Retter et

al. (297) detected 392 (15%) adenoviruses by EM. Of these392, 216 (55%) grew in HAE-70 cells, a continuous line ofhuman amnion cells, whereas 176 (45%) did not produceCPE. However, by immunofluorescence of the non-CPE-producing strains, only isolated cells fluoresced, with nospread to adjacent cells, indicating an abortive infection.Yolken et al. (409), in Baltimore, Md., and Washington,

D.C., found fastidious adenoviruses in 14 of 27 (51.9%) casesof diarrhea during a 12-week period and one fastidiousadenovirus in 1 of 72 (1.4%) children without diarrhea.Although nonfastidious adenoviruses were found in stools of2 of 27 (7.4%) diarrheal patients, such virus also occurred in5 of 72 (6.9%) controls. Fastidious adenoviruses were iden-tified by their growth in 293 cells and by using rabbitantiserum specific for fastidious adenoviruses in a type-specific EIA.

In a 2-year period in Glasgow, 159 stools from 71 childrenunder 3 years of age were found to contain adenovirus byEM in a study by Kidd et al. (195). Established adenovirustypes (1 to 39) were isolated from 81 of the stools from 40 ofthe children, 7 of whom shed two to three adenovirus typessimultaneously. Thirty-six children shed fastidious adenovi-ruses in 64 specimens, and nine of these patients shed bothfastidious adenoviruses and known serotypes at differenttimes. The fastidious viruses produced little or no CPE inroutine cultures or could not be typed or both; they wereidentified by CPE in Chang's conjunctival cells and byneutralization tests with antiserum produced against fastid-ious adenovirus.

It was proposed by de Jong et al. (77) that two antigeni-cally related, fastidious adenovirus variants that had norelationship to the 39 known human adenovirus species becalled adenovirus types 40 and 41. De Jong's group studied200 of these fastidious adenoviruses, which failed to repli-cate serially in conventional human embryonic fibroblast andHEK cells, but many of which could be established inGraham's 293 cells, Chang's conjunctival cells, or sublinesof HeLa or cynomolgus monkey kidney cells that weretreated in special ways. None of the 200 were related totypes 1 to 39 by either neutralization or HAI tests. Thevariants were identical in HAI tests, but DNA restrictionenzyme analyses showed that the two species, 40 and 41,had considerably different genomes. The reference strainsare Tak and Dugan, respectively. Fastidious adenovirusesare ubiquitous with worldwide incidence.From the above studies, it can be seen that fastidious

adenoviruses have been found in many parts of the world.They are probably the second most important cause ofinfantile gastroenteritis after rotavirus. Adenoviruses werefound to be the second most common enteric virus detectedby EM in a number of studies from the United States, UnitedKingdom, Scandinavia, and South Africa (30, 32, 33, 110,185, 198, 324, 367). It was the third most common virus inother studies from the United States (284, 302), Canada(253), India (235), and South Africa (89) and the fourth mostcommon virus in a Scottish study (232), although none weredetected in one South African study (322).

In many instances, fastidious adenovirus infections mayoccur throughout the year with no seasonal variation, ac-cording to reports from the United States, Canada, andScotland (30, 31, 195, 253, 302), although there is a tendencytoward more cases in the warmer months in reports from theUnited States (33, 185), United Kingdom (108), Scandinavia(381), Japan (51), and South Africa (89, 198, 324). Fastidiousadenovirus infection tends to be endemic, rather than epi-demic, although outbreaks in hospital nurseries have oc-

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70 CHRISTENSEN

curred. Fastidious adenoviruses usually infect and causesymptoms in children up to the age of 3 years, but primarilyin those 2 years or younger (110, 195, 381).

Clinical Features

The symptoms associated with fastidious adenovirus in-fection have been studied by a number of investigators.Diarrhea appears to be the predominant symptom, with (195,300, 370) or without (110) vomiting. The severity has rangedfrom mild, afebrile illness (110) to a fatal or "comatosestate" (395) due primarily to dehydration. Reports of theduration of symptoms have been 2 to 4 days (110), a mean of5 days (324), a mean of 7 days (300), and 4 to 8 days (195). Ina Swedish study (370), diarrhea lasted 4 to 23 days with amean of 9 days for those infected with type 40 and 2 to 47days with a mean of 12 days for those with type 41 infection,with one-third of the type 41 patients having symptoms for14 days or longer. Adenovirus-infected patients have milderdisease with less pronounced vomiting and less fever anddiarrhea than rotavirus-infected patients (367).

Respiratory symptoms have also been associated withgastroenteritis in which adenoviruses have been detected inthe patients' stools. In one study (381), clinical signs ofrespiratory tract infection was present in 31% of patientswith diarrhea in which adenovirus was detected in theirstools. In this study, adenovirus was detected by a RIA testin which the antisera used were prepared against a type 2hexon antigen, so that the group antigen was detected. Instill another study (409), the stools of 14 of 27 patients withdiarrhea were found to contain the common hexon antigen ofadenovirus, using an EIA test. Of these 14 patients, 13 alsohad respiratory symptoms such as cough, wheezing, orrhinorrhea; 6 of these had X-ray evidence of pneumonia; and3 had bilateral conjunctivitis. Thus, it was concluded that theenteric types not only cause acute gastrointestinal disease,but also can be associated with a high rate of respiratorydisease.The role of fastidious adenoviruses (types 40 and 41) in

causing respiratory symptoms and that of respiratory ade-noviruses (lower-numbered types) in causing diarrhea havebeen of great interest, but also a problem for the interpreta-tion of data. In some of the earlier studies, adenoviruseswere detected in stools by EM or by EIA or RIA tests withantisera to an adenovirus group antigen. Thus, one cannotdetermine in which outbreaks or with what incidence fastid-ious adenoviruses were found and what disease was attrib-utable to lower-numbered, nonfastidious types. In addition,the term enteric refers only to adenoviruses detected instools, without indicating their fastidiousness or type. Thus,it has been recommended that the term fastidious refer onlyto those adenoviruses that cannot be propagated in routinecell culture or are typed as 40 or 41 or both (228).With techniques becoming available to differentiate be-

tween the fastidious and nonfastidious adenoviruses instools, the role of both groups in causing respiratory andgastrointestinal symptoms could be more thoroughly evalu-ated. In one study by Uhnoo (370), 21% of the patients withadenovirus types 40 and 41 also had respiratory symptoms,i.e., tonsillitis, pharyngitis, otitis, coryza, or cough. Patientswho had diarrhea due to "established" adenovirus types,i.e., types 1 to 39, had diarrhea of shorter duration andhigher fevers, and 79% had respiratory symptoms.

Similarly, Leite et al. (215) detected adenovirus in stoolsof 39 of 746 children with gastroenteritis by IEM or EIA orboth. Of these 39, 25 could be propagated in HEp-2 cells and

neutralized by one of the antisera to types 1 to 18. Theremaining 14 of the 39 could be propagated only in special293 cells and were not neutralized by antisera to types 1 to31. Thus, they concluded that "respiratory" adenovirusescould probably be responsible for gastroenteritis. Sincefastidious adenoviruses have been associated with respira-tory symptoms in some reports, the evaluation of the pres-ence of adenoviruses in stools is a difficult diagnostic di-lemma (215). However, in another study, Uhnoo et al. (367)found respiratory symptoms to occur only rarely in patientsinfected with types 40 and 41 and concluded that theseadenoviruses were restricted to the intestinal tract.Other problems with interpretation of adenoviruses ob-

served in diarrheal stools include (i) long-term shedding ofadenoviruses in the stool and (ii) simultaneous infection withdifferent enteric and respiratory tract adenoviruses. Forexample, Fox et al. (117) found that adenoviruses 1, 2, and 5could be excreted from the intestinal tract without symptomsfor at least 2 years after a primary infection. Other investi-gators have also found that types 1 and 2 especially may beshed in the stools for many months (195).Kidd et al. (195), in a 2-year study, obtained stool samples

containing adenoviruses from 71 children, and serial sampleswere obtained from 35 of these children. Adenoviruses weredetected by EM and by routine cell culture. However, therewas no certainty that an adenovirus seen by EM was the onethat grew in routine cell culture. The longest interval inwhich the same serotype (type 2) was isolated was 231 days.The investigators found that adenoviruses of different estab-lished serotypes may be shed by one child in succession orsimultaneously over a period of days. Four children shedtwo adenovirus types and three children shed three adeno-virus types over a period of 1 to 5 months. In two of thesecases, two types were isolated from the same stool. Thus,dual and triple infections are probably common in children,with prolonged and overlapping infection by different se-rotypes. Of the 71 children, at least 36 shed fastidiousadenovirus, and 9 of these children shed fastidious adeno-virus and known lower serotypes at different times. Al-though common established serotypes were shed over sev-eral weeks or more, excretion of the fastidious adenoviruseswas not seen for longer than 8 days, at least in the amountneeded to be observed by EM.

Brandt et al. (29) addressed the problem of simultaneousinfections with different enteric and respiratory tract viruses.Infants and young children in whom adenovirus or rotaviruswas visualized in their stools were tested for the simulta-neous presence of respiratory viral pathogens in their respi-ratory tracts. Nearly 11% had dual respiratory and entericviral infections. Non-adenovirus respiratory tract pathogenswere seen in 8.5% (4 of 47) of gastroenteritis inpatients, 50%(8 of 16) of respiratory inpatients, and 19% (12 of 63) of totalinpatients with visualized fecal adenovirus. Respiratorysyncytial virus was the most common respiratory tractpathogen isolated from these dually infected patients, fol-lowed by parainfluenza virus type 3.To summarize, in cases of gastroenteritis, it is probably

important to use a diagnostic test that specifically detectstypes 40 and 41 per se. Since a type 40- and 41-specific EIAis now commercially available (Adenoclone-Type 40/41;Cambridge BioScience, Worcester, Mass.), this test couldbe used in conjunction with one of the commercially avail-able rotavirus tests on stool specimens from cases of gas-troenteritis.



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Laboratory Diagnosis

There are a number of methods for detecting adenovirusesand adenoviral antigen or DNA in stools. Some methods areless specific, e.g., EM and some RIAs and EIAs, in thatdifferentiation between fastidious and nonfastidious adeno-viruses cannot be done. Other tests, including type-specificETAs and some hybridization techniques, are specific fortypes 40 and 41. In addition, special cell types can be utilizedto propagate the fastidious adenoviruses.

Initially adenoviruses were detected in stools by routinecell culture (nonfastidious types) and by direct EM (bothfastidious and nonfastidious types). In earlier studies, ade-noviruses in stool extracts could also be detected by coun-terimmunoelectrophoresis (288) and immunoelectroosmo-phoresis (174). RIAs and ETAs to detect adenovirus in stoolshave also been described. Halonen et al. (141) described ahighly sensitive and specific four-layer RIA. In this test,adenovirus type 2 hexon antigen was used as the immunizingantigen for producing hyperimmune sera. The same group(319) also described a similar four-layer EIA.Johansson et al. (175) described two EIAs for adenovirus,

one group specific and one type specific for subgroup F, type40 adenovirus only. The group-specific test detected 35established adenovirus types plus the subgroup F adeno-virus. In both tests, the capture antibody was a group-specific antibody, whereas the detector antibody was eithergroup specific or type 40 specific, depending on the test.Thus by the use of both tests, all adenovirus types in thestool could be detected by the first test, and those of type 40could be differentiated by the second test. In addition,Johansson et al. (176) described a similar type-specific EIAfor adenovirus type 41.

Singh-Naz and Naz (333) developed monoclonal antibod-ies to types 40 and 41 for use in an EIA. Two of thesemonoclonal antibodies recognized a single antigen of 17,000molecular weight, which is probably polypeptide VII, theDNA core polypeptide. One of the two monoclonal antibod-ies reacted with both types 40 and 41, while the other reactedonly with type 40, probably due to reaction with differentantigenic determinants. These antibodies confirmed theclose antigenic relationship as well as the distinctiveness oftypes 40 and 41. Type-specific ElAs for types 40 and 41 arealso commercially available (Cambridge BioScience).

Hierholzer et al. (153) described a monoclonal time-resolved fluoroimmunoassay, using a monoclonal antibodyto adenovirus type 3 hexon antigen. This test was moresensitive than an all-monoclonal biotin-avidin EIA and apolyclonal capture biotin-avidin EIA, but it did not differen-tiate types 40 and 41 from other adenovirus types in speci-mens. However, Wood and Bailey (398) were able to detecttypes 40 and 41 specifically in stool by IEM, using hyperim-mune rabbit sera to these two types.

In addition to these serology-based tests, nucleic acidhybridization tests have been described recently. Stal-handske et al. (343) used nucleic acid extracted from 18RIA-positive stool specimens which were spotted on nitro-cellulose filters (spot hybridization). These were analyzedwith 32P-labeled probes to type 2 DNA and to a clonedBamHI G fragment of adenovirus type 41. Fifteen of the 18stools were positive to the type 2 probe. Five of the 18 werepositive to the type 41 probe and were detected by the type2 probe as well. Thus, by the use of both tests, fastidiousadenoviruses could be distinguished from nonfastidiousones.

Takiffet al. (352) described a similar dot blot hybridization

test, using extracted stool specimens dotted onto nitrocellu-lose and hybridized with 32P-labeled probes to EcoRI andBglII fragments of types 40 and 41. This test was quitespecific and sensitive, detecting as little as 20 pg of adeno-viral 40 and 41 DNA.Kidd et al. (196) described a similar dot blot test, using

only clarified or extracted stool suspensions and hybridiza-tion with 32P-labeled probes to cloned PstI fragments oftypes 40 and 41. The test was relatively fast, taking 48 h.Hammond et al. (143) simplified the hybridization detectiontest by spotting samples directly from stool specimens ratherthan extracting the stools. Niel et al. (271) described a type40 and 41 hybridization test, using a nonradioactive peroxi-dase-labeled probe.

Several of the detection tests described above were devel-oped because fastidious adenovirus types 40 and 41 fromstools could not be propagated in routine cell cultures in thelaboratory. However, Takiff et al. (353) found that thesefastidious adenoviruses could be propagated in Graham's293 cells (128), HEK cells that were transformed by expos-ing the cells to sheared fragments of adenovirus type 5 DNA(128).

In routine cell types, such as HeLa cells, the in vitroreplication of fastidious adenoviruses is probably blocked ata very early stage in the viral growth cycle, whereas thisblock does not occur in the 293 cells (353). In 293 cellsinoculated with stools containing fastidious adenovirus,small foci of progressive adenovirus-like CPE appear afterseveral days (353). The specificity of the CPE can bedetermined by direct fluorescent-antibody test (353). How-ever, the pattern of granular nuclear and cytoplasmic fluo-rescence differs from the fluorescent pattern seen withlower-numbered adenoviruses (353). Fastidious adenovi-ruses grow better when they are seeded into 293 cells thatare subconfluent (36). Graham's 293 cells have also beenused in suspension culture for isolating fastidious adenovi-ruses (329).One problem with the use of 293 cells is that lower-

numbered non-fastidious adenoviruses also grow well inthem (35, 36). In one study by Brown et al. (35), 4 of 15stools positive for type 40 adenovirus also contained alower-numbered adenovirus type, and these lower-num-bered types overgrew the type 40 strain.

In addition to the 293 cells, Kidd and Madeley (197)reported that 42% of fastidious adenoviruses from stoolscould be propagated in Chang's human conjunctival cell line,with development of typical adenoviral CPE. However, notall fastidious adenoviruses could be propagated in this celltype.

ImmunologyAlthough extensive immunologic studies have been car-

ried out on lower-numbered adenoviruses, there have beenonly a few reports on the immunity to adenoviruses 40 and41 per se. Kidd et al. (193) obtained 377 serum samples fromchildren under the age of 12 years living in the UnitedKingdom, New Zealand, Hong Kong, Guatemala, Gambia,and Kuwait. By a neutralization test, they found that at least33% of the sera from the United Kingdom, Hong Kong, andthe isolated country of Gambia had neutralizing antibody to40 and 41. Sixty percent of the New Zealand sera waspositive. The Kuwaiti sera had a lower percentage (15%) ofpositive sera, while all of the Guatemalan sera were nega-tive.

In a study of the age distribution of antibody in Japan,Shinozaki et al. (328) found that 20% of 1 to 6 month olds and

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50% of 37 to 48 month olds were antibody positive to 40 and41 by neutralization tests. Of adults, 48% of 18 to 20 yearolds and 10% of people over 70 years of age were type 40 and41 positive. All of the sera were positive to the adenoviruscommon antigen.

Prevention and Treatment

Experimental vaccines to types 40 and 41 have not yetbeen developed. Problems involve the relatively poor repli-cation of fastidious adenoviruses in cell culture. The use of293 cells as a substrate would be inappropriate, since theseare transformed cells. In addition, both types 40 and 41 havebeen shown to have transforming ability in baby rat kidneycells (374). Fastidious adenovirus infection is not as commonor as serious as rotavirus infection, and therefore the needfor an adenovirus vaccine is not as pressing as that for arotavirus vaccine. Treatment includes maintaining properelectrolyte balance and fluid levels, as in rotavirus infection.

NORWALK AND NORWALK-LIKE VIRUSES

Introduction

Whereas rotavirus primarily causes disease in infants andyoung children, Norwalk and Norwalk-like viruses primarilyinfect and cause disease in older children and adults. TheNorwalk virus is a small, round virus, 25 to 30 nm indiameter, which was first detected by Kapikian et al. (187)by EM examination of stools of patients from an outbreak ofwinter vomiting disease occurring in Norwalk, Ohio, in 1968.Several other viruses resemble Norwalk virus by theirmorphology, the type of clinical illness they cause, and theirlow concentration in infected stools. However, some areantigenically similar and others are antigenically distinctfrom the Norwalk agent. These other agents, discovered inthe 1970s, include the Hawaii agent (401), the MontgomeryCounty agent (356), discovered in Maryland, and the SnowMountain (SM) agent, discovered in Colorado (87). Theseviruses, including the Norwalk agent, cannot be cultivated invitro, so that little is known of their biophysical and bio-chemical characteristics. The viruses can be propagated onlyin human volunteers and in chimpanzees (133).

Since very small amounts of these viruses are excreted inthe stools of infected persons, the viruses can be detected byIEM, using convalescent sera from recovering patients.Frequently, they may not be detected at all. In such cases, adiagnosis may be made by detecting a significant serologicrise in antibody titer.Other small, round viruses which were originally thought

to be Norwalk-like viruses are now considered to be candi-date parvoviruses and include the Cockle agent and theDitchling agent (6, 42).

Physical Characteristics

Norwalk and Norwalk-like agents observed by EM orIEM are 27 to 30 nm in diameter and have been described aspicorna/parvovirus-like, as well as resembling caliciviruses(described below). However, their fine morphology is diffi-cult to distinguish because of their small size and because,by IEM, the virions are coated with antibody. Since theseagents cannot be cultivated in vitro, biophysical and bio-chemical studies have been carried out on virus purified fromdiarrheal stools of infected patients.The Norwalk agent is resistant to ether, since it lacks a

lipid-containing outer envelope (22, 84). The Norwalk agent

is relatively resistant to acid (pH 2.7 at room temperature),and this characteristic may protect the agent in its passagethrough the stomach (22, 84). The Norwalk agent is alsorelatively heat stable; after a viral suspension is heated to60'C for 30 min, the suspension can still produce disease insome volunteers (22, 84).Norwalk virions, purified by Greenberg et al., appear to

contain a single primary structural protein with a molecularweight of 59,000 (133). In addition, this group detected asoluble viral protein with a molecular weight of 30,000 in thestools of Norwalk-infected patients. The protein structure ofthe virion is similar to that of the Caliciviridae, which hasone major structural protein of 60,000 to 71,000 molecularweight (133, 233). Parvoviruses differ in that they have threedifferent structural proteins (133). The Norwalk agent has abuoyant density in CsCl of 1.38 to 1.40 g/cm3. The Calicivi-ridae size (35 to 40 nm) and buoyant density in CsCl (1.36 to1.39 g/cm3) are similar, though not identical, to those of theNorwalk agent. The Hawaii agent has a density in the rangeof 1.38 to 1.40 g/cm3 in CsCl (356).The SM agent has been studied more extensively and

compared with feline calicivirus (233). In CsCl, the SM agentpeaked at 1.34 g/cm3 with a shoulder at 1.37 g/cm3 comparedwith the calicivirus, which peaked at 1.37 g/cm3 with asmaller peak at 1.33 g/cm3. On potassium tartrate-glycerolgradients, SM virions of two densities, 1.21 to 1.22 and 1.29g/cm3, were observed, whereas the calicivirus peaked at 1.29g/cm3 with a smaller peak at 1.21 g/cm3. In analyzingvirion-associated proteins, a major SM agent structuralpolypeptide of 62,000 molecular weight was observed, sim-ilar in molecular weight to the major calicivirus structuralprotein of 65,000. These results suggested that SM, likeNorwalk agent, has properties resembling that of the calici-virus group and may suggest a relationship between the twogroups. The buoyant densities of Hawaii agent were found tobe 1.37 to 1.39 g/cm3, with most virions recovered at 1.38g/cm3 (356). The buoyant densities of the MontgomeryCounty agent complete virions had a range of 1.37 to 1.41 g/cm3, with the majority of virions found at 1.39 g/cm3 (356).Empty particles were seen at 1.30 g/cm3 (356).

Epidemiology

Norwalk virus has been responsible for a large number ofoutbreaks of acute infectious nonbacterial gastroenteritis. Inone report, Greenberg et al. (136) obtained paired sera frompatients in 25 outbreaks of gastroenteritis occurring over a12-year period. Using the sera in a RIA test, they determinedthat 32% (8 of 25) of outbreaks were due to Norwalk orantigenically related viruses. One of the outbreaks was dueto rotavirus, but the causes of the remaining outbreaks inthis study could not be determined. The Norwalk outbreaksoccurred in a grade school, colleges, a family, and cruiseships at sea. In a more extensive study by Greenberg et al.(137), 34% of 70 outbreaks of gastroenteritis were associatedwith Norwalk or antigenically related agents. In still anotherstudy, Kaplan et al. (189) reported that 38 of 81 outbreaks ofgastroenteritis were caused by Norwalk or antigenicallyrelated viruses and gave detailed information on these oc-currences. Ten outbreaks occurred in camps and recre-ational areas; seven, in elementary schools; four, in colleges;three, in small families; two, in larger communities; four, inrestaurants; four, in nursing homes; and four, on ships atsea. Three occurred in other countries, while the remainderoccurred in the United States. In this last study, the sourcesof infections included municipal water systems in two out-



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breaks, semipublic water supplies in seven, recreationalswimming in two, and stored water on cruise ships in two;eating contaminated oysters in two and eating salad in two;and primary person-to-person transmission in seven. Inaddition, secondary person-to-person transmission occurredin a number of the outbreaks. The outbreaks lasted anaverage of 7 days, with a range of 1 day to 3 months. Inseven outbreaks, successive weekly outbreaks occurredamong newly introduced populations as on cruise ships or atcamps. The largest outbreaks occurred in communities,schools, recreational areas, and on cruise ships, with anaverage of 348 people involved and with a range of 19 to2,000 cases. Smallest outbreaks occurred in families andnursing homes. One nationwide outbreak was associatedwith eating raw oysters that were shipped around the coun-try. The epidemiologic and clinical characteristics of thenon-Norwalk outbreaks suggest that they were caused byagents resembling, but antigenically distinct from, the Nor-walk agent. They were similar in their incubation period, theprevalence of symptoms, and the duration of illness. In twoof these outbreaks, 27-nm particles resembling Norwalkvirus were seen in the stools of affected patients.Although the role of Norwalk virus and antigenically

related viruses in large and small outbreaks of gastroenteritisis well documented, little is known about their endemic rolein disease causation. This is due in part because patientsusually are not hospitalized, and routine diagnostic labora-tories do not have the reagents available for detecting theseviruses or viral antibodies.

Since Norwalk and similar viruses tend to infect olderchildren and adults in developed nations, one would expectthat these older populations would have a higher prevalenceof antibodies than in small children. This is in contrast torotavirus, in which antibody development occurs early inlife, due to early infection with this virus. Several studieshave addressed the issue of the development of antibody toNorwalk virus throughout different age groups which wouldreflect the acquisition of Norwalk infection. Blacklow et al.(21) found that acquisition of antibody to Norwalk virus inAmerican subjects was minimal during childhood and was inthe range of only 5% before the age of 12 years. Throughoutadolescence and early adulthood, the prevalence of antibodyrose rapidly, reaching 45% in 18 to 35 year olds and reachingto 55 to 60% in 45 to 65 year olds, before declining slowly inthe elderly.

In studying different populations, Greenberg et al. (134)found that antibody acquisition to Norwalk virus occurredmuch earlier in less developed nations. In underdevelopedcountries such as Bangladesh and Ecuador, antibody levelsranged from 75 to 100% throughout childhood, while inYugoslavia the rate of acquisition was between that of theless developed countries and that of the United States.Cukor et al. (72) found the rates of acquisition of antibody toNorwalk virus to be slow in the United States and in Taiwan,similar to Greenberg's rates, with rates in the Philippineshigher, approaching that found in Yugoslavia by Greenberget al.The report of Greenberg et al. (134) also noted the

ubiquitousness of antibody to Norwalk virus from aroundthe world. They looked at antibody levels in blood obtainedfrom volunteer blood donors and found that 54 to 77% ofblood from urban United States, Belgium, Switzerland, andYugoslavia and from rural Bangladesh and Nepal had anti-body to Norwalk virus.

Clinical Features and Pathogenesis

Clinical features. The clinical features of the Norwalk andsimilar agents have been reported from both the originaloutbreaks of these viruses and from studies in volunteers.The infections produced by Norwalk virus (85), the Hawaiiagent (356), the Montgomery County agent (356), and theSM agent (87) are similar. The incubation periods wereusually 24 to 48 h, with sudden onset. The most predominantfeatures were nausea and vomiting, often severe. Low-gradefever and diarrhea usually occurred, but the latter tended tobe relatively mild. Diarrheal stools did not contain blood,mucus, or leukocytes (86). Other symptoms included mod-erate to mild abdominal pain or cramps, headache, andmalaise. Laboratory tests were within normal limits involunteers inoculated with Norwalk agents, except that onehad a transient elevation of leukocytes. The attack rate withthese agents may be high, affecting 50% or more of themembers of a school, camp, or other institution or groupwhere an outbreak occurs. Respiratory symptoms do notappear to be a manifestation of Norwalk or Norwalk-likeinfections, which differs from that of rotavirus and possiblyadenovirus infection.

Children are more likely to have vomiting, whereas adultsare more likely to experience diarrhea (189). The vomitingmay be caused by a decrease in gastric mobility, which maybe more pronounced in children than in adults (189).Although the disease seldom requires hospitalization, se-

rious illness has occurred in rare instances. Hospitalizationof a total of three middle-aged persons for severe dehydra-tion was reported in two outbreaks (189). In one nursinghome outbreak, three patients needed intravenous fluids, butthey were not hospitalized. In another nursing home out-break, two elderly debilitated patients died after the onset ofgastroenteritis, but supposedly the deaths were due to dif-fuse atherosclerosis (189).

Pathogenesis. The amount of virus needed to initiate aninfection in human volunteers has been estimated. In onestudy, the Norwalk agent had a titer of at least 500 humandisease-producing doses in the first human passage filtrate,whereas the disease was produced with only undiluted stoolfiltrate from a second human passage (22). In a study withthe SM agent, Dolin et al. (87) reported that five of sixvolunteers given 1.0 to 0.5 ml of undiluted stool suspensionbecame ill, two of two volunteers given 1.0 ml of a 1:10dilution became ill, two of two given a 1:100 dilution becameill, and none given a 1:1,000 dilution became sick.

Evaluating the respiratory route as a means of transmis-sion of Norwalk infection, Blacklow et al. reported thatnasopharyngeal washings from a volunteer acutely ill withNorwalk infection failed to induce disease in other volun-teers (22). Thus, they concluded that the respiratory route isnot a major route by which this virus could be transmitted.

Jejunal biopsies from young adult volunteers obtainedbefore, during, and after administration of suspensions ofNorwalk virus have been described by Agus et al. (2) asfollows: by light microscopy, abnormalities in the intestinalmucosa at the height of illness included partial villousflattening, broadening of the villi, and disorganization of theepithelial lining cells. Moderate infiltration of the laminapropria by mononuclear cells also occurred, and there werefocal areas of epithelial vacuolization. EM of the jejunalepithelial cells showed dilation of the rough and smoothendoplasmic reticulum and an increase in multivesiculatebodies. The microvilli were shortened, and the intercellularspaces were widened and filled with an amorphous electron-

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dense material. No definite viral particles were seen. Con-valescence biopsies carried out 2 weeks later showed thatthe mucosa had returned to normal in all volunteers.Agus et al. (2) also showed that, in addition, jejunal brush

border enzyme activities, including alkaline phosphatase,sucrase, and trehalase levels, were decreased. Convales-cence enzyme levels determined 2 weeks later returned tothe base-line rates in all volunteers. These reduced enzymelevels may contribute to the pathogenesis of Norwalk dis-ease. However, in contrast to the intestinal lesions seen withinvasive bacterial agents, such as Shigella spp. and entero-pathogenic Escherichia coli, in Norwalk-induced disease themucosa remained intact. Rectal biopsies taken at the heightof illness showed a normal histologic pattern. There was anabsence of fecal leukocytes in Norwalk-induced illness.Thus, the colonic mucosa is relatively spared in this syn-drome as compared with bacterial gastroenteritis. Inoculatedvolunteers who were asymptomatic had normal jejunal bi-opsies.

Schreiber et al. (325) described the pathologic lesions seenin volunteers who ingested stool filtrates containing theHawaii agent. The volunteers developed intestinal mucosallesions in the proximal small intestine that were similar tothose lesions seen in Norwalk infection. However, these twoagents are immunologically distinct. Lesions from sympto-matic volunteers showed shortening of the villi and anincreased cellularity of the lamina propria. Many villousabsorptive cells were decreased in height, and some hadvacuolated cytoplasm. Increased numbers of mononuclearcells and some polymorphonuclear leukocytes infiltrated theintercellular spaces between epithelial cells. Many polymor-phonuclear leukocytes and increased numbers of mononu-clear cells were seen throughout the lamina propria. Two offour volunteers without symptoms developed mucosal le-sions indistinguishable from those of sick volunteers 48 hafter ingestion of virus. The remaining two of four asympto-matic volunteers had normal mucosal epithelia (325). Dolinet al. (86) also described jejunal biopsies from Hawaiiagent-infected volunteers and found that they were similar,but not identical, to those seen in Norwalk virus infection.Light microscopy of biopsy specimens showed an intactmucosa, moderately blunted villi, and a moderate inflamma-tory cell infiltrate in the lamina propria, consisting of bothpolymorphonuclear and mononuclear cells. EM showeddistorted and markedly shortened microvilli on intact epithe-lial cells, enlarged pale mitochondria containing indistinctmembranes, and moderate widening of intercellular spacesfilled with an amorphous electron-dense material. The biop-sies of the asymptomatic volunteers remained normal. By 2to 3 weeks postinfection, the lesions had disappeared.

Laboratory Diagnosis

The laboratory diagnosis of Norwalk and similar agentshas been hindered by the inability to propagate these virusesin vitro. Reagents are available in research laboratories, butare not available for routine diagnostic laboratories. Inaddition, routine laboratory diagnosis of these agents is notcarried out since patients do not usually require hospitaliza-tion, where testing is usually done.

Cell and organ culture. Attempts at propagating the Nor-walk agent in a number of types of cell cultures has failed.However, its replication has been attempted in organ cul-tures of human fetal intestine (22, 84). Full-thickness ex-plants approximately 2 mm2 in size can be maintained in thelaboratory for up to 3 weeks. They have differentiated cells

and tissues, including villi, that maintain their morphologicintegrity and their in vivo relationship to each other. Inocu-lation of Norwalk-containing stool suspensions into theseorgan cultures did not cause any morphologic alteration inthe cultures that were seen by light or dissecting micros-copy. However, fluids from the organ cultures ingested byvolunteers did cause illness in some volunteers. Whether ornot the virus actually replicated, or whether virus from theoriginal inoculum was responsible for the illness, was notknown.

In vitro assays. The first assay used to detect Norwalk andNorwalk-like viruses, and to semiquantitate antibodies tothem, was IEM. By this method, virus could be detected instool suspensions by mixing a small amount of the suspen-sion with sera from patients recently recovering from Nor-walk infection. After placing the mixture on a grid andnegatively staining with phosphotungstic acid, clumped Nor-walk particles could be visualized. Similarly, known Nor-walk-containing suspensions could be used to detect anti-bodies in sera of patients recovering from gastroenteritis.This technique was useful in initially detecting these virusesand studying the development of immunity. However, forextensive epidemiologic and other studies, in which largenumbers of samples are examined, it was somewhat slowand cumbersome. Compared with tests developed later,IEM also lacked sensitivity.

Since the Norwalk and Norwalk-like viruses cannot bepropagated in vitro, investigators developing in vitro assayshave had to rely on patient stool specimens for sources ofvirus and their convalescent sera for sources of antibody.Virus from stool samples cannot be purified in sufficientquantity to produce hyperimmune animal sera or monoclo-nal antibodies. Because of the lack of reagents, assays havebeen developed only in a few research laboratories, and noreagents are available commercially. The few assays thathave been developed are the RIA, the EIA, and the biotin-avidin-EIA.

Greenberg et al. (138) developed two similar solid-phaseRIAs for detecting either Norwalk viral antigen or antibody.In both assays, patient convalescent serum was used as acapture antibody and acute-phase serum was used as acontrol antibody to coat microtiter plate wells. To use theRIA to detect viral antigen, suspect stool suspensions wereadded to both acute- and convalescent-phase serum-coatedwells. This was followed by the addition of detector antibodyconsisting of IgG purified from convalescent-phase serumthat was labeled with 1251.To detect the presence of antibody in patient sera, Green-

berg et al. (138) used a blocking RIA. In this test a stoolsuspension known to contain Norwalk virus was added tothe capture-antibody-coated wells. The patient sera to betested for Norwalk antibody were then added. Lastly, a125I-labeled detector antibody was added. A similar RIA forNorwalk virus has been described by Blacklow et al. (21).Dolin et al. (88) have described a similar RIA for the SMagent. These RIAs were shown to be highly specific andsensitive. However, other problems have been associatedwith them. The primary problem has been the rapid loss ofreactivity of the 125I-labeled detector antibody, which isgood for <1 week after labeling. In addition, there are theusual costs, hazards, and disposal problems associated withthe use of radioactive substances.

Because of the inherent problems with the RIA, an EIAfor Norwalk antigen detection has been developed (149,150). This test is similar to the RIAs. Again, the reagentswere human clinical materials and differed only in that the



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IgG fraction from convalescent serum used as detectorantibody was labeled with horseradish peroxidase ratherthan 125I. The major advantage of the EIA is the stability ofthe detector reagent. In addition, the EIA's ability to detectviral antigen appeared to be slightly more sensitive than thatof the RIA. The test was quite sensitive in that there was nocross-reactivity between the two antigenically distinct Nor-walk and Hawaii agents.Two biotin-avidin ElAs have been described that detect

Norwalk or SM antigen or antibodies (120, 234). These twoagents have been shown to be antigenically distinct. TheseEIAs are similar to the RIAs and EIA described except thatthe IgG detector antibody is labeled with biotin, rather thanperoxidase or 1251. In the final steps of the tests, avidin-peroxidase, an enzyme substrate, H202, and a chromogenare added as an indicator. These EIAs were as sensitive ormore sensitive than comparable RIAs from these laborato-ries for the detection of antigen. All of these tests have beenused in the epidemiologic and immunologic studies reportedin this treatise.

Immunology

Since individuals have repeated episodes of gastroenter-itis, it was of interest to determine the length of time oneremains immune after infection with the Norwalk agent orsimilar viruses. It was also of interest to determine one'scross-immunity to Norwalk-like viruses and to determine theantigenic relationships among the various Norwalk-like vi-ruses. First, to study homologous immunity to Norwalkvirus, Blacklow et al. (22) reported infecting volunteers withNorwalk agent and rechallenging these volunteers 6 to 14weeks later. None became ill, indicating that at least short-term homologous immunity occurred after Norwalk virusinfection. Similarly, Wyatt et al. (401) found immunity involunteers when they were rechallenged 9 to 14 weeks afterexperimental infection with Norwalk agent. In addition,when volunteers were infected with the Hawaii agent andchallenged 6 to 7 weeks later with the same agent, nonebecame ill. When volunteers were infected with the Mont-gomery County agent and challenged 7 weeks later with thehomologous virus, none became ill. This indicated at leastshort-term homologous immunity for these three viruses.Heterologous challenges by Wyatt et al. (401) were also

carried out. Three of six volunteers given Norwalk agent andchallenged 7 to 15 weeks later with Hawaii agent became ill,indicating that the Norwalk and Hawaii agents were antigen-ically dissimilar. When six volunteers given Norwalk viruswere challenged 6 to 13 weeks later with the MontgomeryCounty agent, none became ill, but the reverse was not true.One of three volunteers given Montgomery County agentand challenged 11 to 12 weeks later with Norwalk agentbecame ill. Thus, Norwalk infection appeared to protectagainst Montgomery County agent, although MontgomeryCounty infection may not protect against Norwalk infection.However, when four volunteers given Hawaii agent werechallenged 8 weeks later with Montgomery County agent,none became ill, indicating an antigenic relationship betweenthese two viruses.

Parrino et al. (281) orally inoculated 12 volunteers withNorwalk virus and found that six became ill with vomiting ordiarrhea or both. Sera were collected from five of thevolunteers. By IEM, four of five had initial low-level anti-body which increased after challenge and then declined inthe interim time between the first inoculation and a second,reinoculation 27 to 42 months later. When reinoculated with

Norwalk virus 27 to 42 months later, the same six peoplebecame sick, indicating that immunity to Norwalk agent wasrelatively short-lived. Again, antibody levels increased afterchallenged in the same four individuals. A fifth individualwho became sick each time he was challenged had persis-tently high antibody levels before and after each challenge,indicating that serum antibody alone does not protect againstinfection with Norwalk virus, at least in some individuals.When the volunteers were challenged a third time 4 to 8weeks after the second challenge, all except one remainedwell, indicating that at least some immunity occurs for ashort period of time. The six remaining volunteers did notbecome sick the first time they were inoculated with Nor-walk virus. When rechallenged 31 to 34 months later, theyagain resisted infection. Serum samples obtained from threeof these volunteers had low levels of antibodies detected byIEM which remained low before and after both challenges.From these studies of Parrino et al. (281), there appeared

to be two forms of Norwalk immunity. The first group wassusceptible to virus infection and developed only short-termimmunity. The second group maintained long-term immu-nity, although they showed only low-level antibody to Nor-walk or possible cross-reactivity to Norwalk due to antibod-ies to a related virus. However, protective mechanismsother than serum antibody appeared to protect them againstdisease. It is possible that individuals in the second grouplacked a receptor for Norwalk virus on their intestinalmucosal cells that are usually infected by this virus.

In a similar study, Blacklow et al. (21) found that themajority of volunteers given Norwalk virus who became sickhad preexisting Norwalk antibody, whereas the majority ofvolunteers who did not become ill did not have preexistingNorwalk antibody or had very low levels of antibody. Inaddition, all of those volunteers who had preexisting anti-body in their duodenal fluids developed illness, whereas onlyone-half of those with no duodenal antibody became sick.Thus, clinical immunity to Norwalk virus appears to becomplex. Some individuals appear to be susceptible torepeated infections with Norwalk virus, whereas other indi-viduals appeared to be resistant to Norwalk infection (21).From immunologic studies, the possible relationship of

Norwalk virus to calicivirus is unclear. Madore et al. (234),using a biotin-avidin-EIA, did not detect any cross-reactionbetween feline caliciviruses and sera from Norwalk or SMvirus-infected individuals. However, Nowak et al. (N. A.Nowak, W. D. Cubitt, J. E. Herrmann, and N. E. Blacklow,Abstr. Annu. Meet. Am. Soc. Microbiol. 1986, C73, p. 340)studied 18 paired serum samples from four human calicivirusoutbreaks, using a Norwalk RIA. From two calicivirus typeUK4 outbreaks, 7 of 10 paired sera showed a significantantibody rise to Norwalk virus. The Norwalk antibody titerswere similar to those seen in sera from outbreaks of Norwalkdisease. Thus, the latter data provide evidence for at least aone-way serologic cross-relatedness between Norwalk virusand calicivirus (88).

Prevention and Treatment

Since Norwalk and Norwalk-like viruses cannot be grownin vitro, no vaccines are available. In addition, vaccines maynot be necessary since infections usually occur sporadicallyor in isolated outbreaks and clinical illness is short-lived andrelatively mild in older children and adults who becomeinfected. There are a variety of measures to prevent out-breaks and sporadic infection due to these agents. Theseinclude good personal hygiene to prevent primary and sec-

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ondary person-to-person contact, good personal hygieneamong food handlers, not allowing food handlers with gas-troenteritis to work, and not ingesting raw or undercookedshellfish. In addition, outbreaks can be prevented by propersewage and water treatment, maintenance of proper watersupplies, and avoidance of contamination of water supplieswith sewage systems, particularly in rural or underdevel-oped areas.Treatment includes maintaining electrolyte and fluid levels

in patients.

CALICIVIRUSES

Caliciviruses were known to infect mammals before theywere discovered in humans. They were first described inhumans by Madeley and Cosgrove (231) and Flewett andDavies (112), who found them by EM in the stools frombabies with diarrhea.

Caliciviruses were originally considered to be in the familyPicornaviridae but have now been placed in the new familyCaliciviridae. They are RNA viruses about 30 nm in diame-ter. Caliciviruses derive their name from the 32 cup-shapeddepressions on the surface of their virions. This gives theirperiphery a spiky appearance and causes the formation of aStar of David configuration at certain rotations of the virion(Fig. 1C). The Star of David configuration is formed by sixperipheral hollows surrounding a seventh central hollow(227, 331). The buoyant density of human calicivirus in CsClranges from 1.38 to 1.40 glcm3, with a peak at 1.39 g/cm3(346).

Caliciviruses primarily cause gastroenteritis in infants andyoung children. They can cause gastroenteritis in the generalpediatric population (67, 267, 346) as well as outbreaks ininstitutions including schools (248), orphanages (53, 54), amother-baby psychiatric unit (69), and nosocomially in pe-diatric wards in hospitals (67, 341). Sporadic cases haveoccurred in the United Kingdom, Norway, Australia, Can-ada, United States, India, and Bulgaria (68).Most individuals are infected with caliciviruses at a very

young age. In Madeley and Cosgrove's first report, all buttwo children were 2 months old or younger. In other reports,patients were 2 to 20 and 1 to 27 months old in orphanageoutbreaks (53, 54); 1 to 18 months old in a mother-babypsychiatric unit (69); 6 weeks to 13 years, with a peak at 1 to6 months old in a general hospitalized population (67); and 4to 6 years old in a school outbreak (248). However, in oneoutbreak occurring in a residential home for the elderly, 18affected residents were 77 to 102 years old, and the 32affected staff members were 16 to 68 years old (70).

Calicivirus infections were found to occur throughout theyear, with peaks in the winter months, according to one30-month study (67). Sporadic as well as more epidemicinfection can occur (67).The symptoms of calicivirus infection are about as severe

as those seen with rotavirus infection and may be indistin-guishable from them (67). In two studies, diarrhea wasreported as the predominant feature (341, 346). In one study,infants had either diarrhea only or diarrhea plus vomiting(53). In one 30-month study of hospitalized pediatric patients(67), diarrhea was the most common symptom in 89%, 50%had vomiting in addition to diarrhea (usually in the 4 to 6year olds), one-third had upper respiratory tract symptoms,and about 20% had fever. One patient had projectile vomit-ing (67). In 4 to 6 year olds in a school, most patients hadnausea and vomiting, although some also had diarrhea (248).In initially well babies in the mother-baby psychiatric ward,

some had mild vomiting and diarrhea lasting only 1 day,some had severe vomiting and diarrhea lasting 7 to 9 days,and one had very severe projectile vomiting (69). Twoorphanage outbreaks lasted 4 to 6 days, and viral sheddingwas correlated with the days of illness, which rarely oc-curred for more than 10 days (54). In the 30-month study(67), the illness lasted an average of 4 days, with a range of2 to 11 days. In the initial report of Flewett and Davies (112),calicivirus was found in the small bowel obtained at thenecropsy of a 22-month-old child who died of gastroenteritis,but the investigators were not sure of its significance.Caliciviruses have also been found in the stools of wellindividuals (53, 231), and in the outbreak in babies in themother-baby psychiatric unit, none of the mothers or staffdeveloped symptoms (69). However, in the elderly in theresidential home (70), 100% of the sick residents and staffhad diarrhea and 50% of both groups had vomiting. A few ofthe staff members also reported headaches, general aches,and malaise, although the elderly residents did not.

In the 30-month study of patients hospitalized with gas-troenteritis in the United Kingdom, 6.6% was due to calici-virus (67), while in another 3-year study in Japan, 1.2% wasdue to calicivirus (346). Thus, caliciviruses are responsiblefor only a minority of viral gastroenteritis among hospital-ized patients, although they have worldwide prevalence (seebelow).

In several studies there was good correlation betweenenteric disease and the presence of caliciviruses in stool seenby direct EM (53, 54, 69, 70). In addition, paired acute andconvalescent serum samples were obtained from the pa-tients, and rises in antibody titers to caliciviruses were seenby 1EM (53, 69, 70, 265). Caliciviruses in patient stools wereused as the source of virus for IEM.No one has been able to isolate the human caliciviruses in

routine cell cultures (53, 70, 346), although other mammaliancaliciviruses such as feline calicivirus can readily be propo-gated in cells of their species (231). In addition to EM for thedetection of caliciviruses in stools, a solid-phase RIA hasbeen developed by Nakata et al. (264). They prepared guineapig hyperimmune serum for the test by using as an antigencalicivirus particles purified from patient stools. They foundthe RIA to be much more sensitive than direct EM fordetecting caliciviruses in stools. With a blocking RIA, theirtest was shown to be specific for calicivirus. This RIA is alsoadvantageous in that caliciviruses are smaller and present inlower numbers in stools than rotaviruses and thus are harderto detect by EM. In another report, Nakata et al. (266)described a similar EIA that was as sensitive as their RIA.To determine the prevalence of antibody to calicivirus in

the general population in Japan, Sakuma et al. (312) usedIEM to detect antibody in 83 serum specimens. The age-related prevalence of antibody was 23% in 0 to 5 month olds,30% in 6 to 23 months olds, 65% in 2 to 5 year olds, and 90%in school children and adults. Similar results were found byNakata et al. (266) in sera from healthy children in theUnited States. All (100%) infants 0 to 3 months old hadcalicivirus antibody, whereas the percentage dropped to 25%for 4 to 11 month olds; by the ages of 4 to 6 years, however,100% of children had developed antibody. Thus, calicivirusappears to infect the population early in life.Nakata et al. (263) used their blocking RIA to determine

antibody prevalence in 390 sera from adults in Japan, Indo-nesia, Singapore, and Papua New Guinea. The positivityrates were 87, 88, 70, and 94%, respectively. The resultsindicate that the virus is a common infectious agent in Japanand Southeast Asia.



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A study of the worldwide prevalence of antibody tocalicivirus strain UK1 was carried out by Cubitt and Mc-Swiggen (68). High antibody titers were seen in all batches ofpooled immune globulin from the United Kingdom, Belgium,Switzerland, Canada, United States, and Japan. The per-centage of antibodies in this calicivirus study increased withage in patients from the United Kingdom and Japan and inSaudi Arabian Bedouins. All older children and adolescentsin Tanzania had antibodies; sera from younger children werenot available. The only major difference in antibody preva-lence was seen in Australian aborigines. Here there was littleevidence of infection before the age of 13 years, althoughmost adults had antibody to calicivirus. Thus, the viruscauses infection worldwide, occurring first in young childrenand infants.At least two strains of calicivirus have been recovered

from children. However, the agent responsible for the out-break in the home for the elderly was antigenically distinctfrom the two strains isolated from children (70).To date, vaccines cannot be developed to control calici-

virus infection, since the virus cannot be propagated in vitro.Treatment consists primarily of maintaining the proper elec-trolyte balance and fluid balance, as in rotavirus gastroen-teritis. This is important since calicivirus infection is proba-bly as serious as rotavirus gastroenteritis.

ASTROVIRUSES

Human astroviruses were first discovered by EM in thestools of infants with diarrhea by Madeley and Cosgrove(229, 230). Shortly thereafter, astroviruses were found toinfect various species of mammals, including cattle, sheep,and pigs (209, 400), causing diarrhea, especially in the youngof these species.

Astroviruses have been isolated in the United Kingdom (7,210, 229, 230) Norway (199), Federal Republic of Germany(226), Japan (204), and North America. They are responsiblefor about 5% of infantile gastroenteritis, including noso-comial infections. The Marin County agent, discovered inelderly patients with diarrhea in a convalescent hospital inCalifornia, is also an astrovirus (148, 278).

Their appearance has been described in detail by Madeley(227). They are 28- to 30-nm, round virus particles with asmooth edge, as opposed to caliciviruses which have a roughedge. Most of the virus particles have a star-shaped config-uration on their surface (Fig. 1D). This star configuration canhave either five or six points, whereas caliciviruses haveonly six-pointed stars. The point of the astrovirus starradiates our from a central point or knob and has no hollowcenter as does the calicivirus. The astrovirus buoyant den-sity in cesium chloride has been reported to be 1.39 to 1.40(204) and 1.33 to 1.34 (226) g/cm3.There appear to be at least five distinct serotypes of

human astroviruses (199, 209, 214). These have been distin-guished by an immunofluorescence test and by immunosor-bent EM. In the former test, acetone-fixed astrovirus-in-fected LLC-MK2 or HEK cells were used. In the latter test,astrovirus antisera against the various strains were raised inrabbits and used to coat Formvar-coated EM grids. Theastrovirus suspensions were added to these grids. The gridswere examined and the number of virus particles wascounted. The only cross-reaction seen was with serotypes 1and 3. There were no cross-reactions of any of the fivehuman strains with bovine or ovine astroviruses. Fromstrains collected over a 10-year period in the United King-dom, serotype 1 appears to be the most prevalent, account-

ing for 77% of the astroviruses. The remaining types 2 to 5had a similar incidence, ranging from 5 to 7%.

In determining the incidence of astrovirus infection inJapan, Konno et al. (204) found that 50% of young adultstested by IEM had antibodies to astroviruses. They alsofound astrovirus antibodies in the commercial gamma glob-ulin preparation they tested. By IEM, they found no cross-reaction with caliciviruses, Norwalk virus, polioviruses, or acoxsackievirus B.

Children, from infancy to 5 to 7 years of age, are mostlikely to develop symptomatic disease with astroviruses (7,204, 210), although exposed adults can also develop milddisease, but with less frequency (204, 210, 278). In one study(210), older children were also found to be less likely todevelop disease than younger children. Asymptomatic infec-tion can also occur among infants and young children (7,229).The incubation period appears to be 24 to 36 hours, similar

to those for other gastroenteritis viruses (204). Symptomscan last from 12 h to 3 to 4 days (7, 204, 210).

In one study, all symptomatic children had watery diar-rhea, while only one-third had vomiting (210). However, inanother study, some children had vomiting only, while theremainder suffered from both vomiting and diarrhea (7). In athird study, the rates of symptoms were as follows: vomit-ing, 74%; diarrhea, 30%; abdominal pain, 49%; and fever,30% (204). Clinical illness appeared to be less severe thanwith rotavirus (210).

Astroviruses can be detected in stool suspensions bydirect EM (7, 210, 229, 230). Konno et al. (204) found thatthe star configuration was much more distinct if the particleswere stained with phosphotungstic acid rather than withuranyl acetate.

Astroviruses, including the Marin County agent (278), canbe isolated and propagated in HEK cells to which trypsin-containing medium is added (148, 213, 214). Astrovirus-infected HEK cells can be used for immunofluorescencetests and EIAs for serologic studies (148, 209, 214).

Antibodies can be detected by IEM. Antibody rises can beobserved in paired acute and convalescent sera obtainedfrom patients (204, 210). In addition, astrovirus-specific IgMcan be detected by IEM after fractionation of patient sera bysucrose density gradient centrifugation (7).There is no specific treatment for astrovirus infection,

similar to other viral gastroenteritides. Nonspecific treat-ment consists of maintaining fluids and electrolyte balance,although astrovirus infection appears to be less severe thanrotavirus infection (210).

CORONAVIRUSES

Coronaviruses are enveloped, medium-sized, positive-stranded RNA viruses that were first discovered in patientswith upper respiratory tract illness. These viruses have alsobeen associated with diarrheal diseases in mammals, espe-cially newborns. They may be involved in human gastroin-testinal disease (296), since by direct EM coronavirus-likeparticles (CVLP) have been seen in stools of patients withacute nonbacterial gastroenteritis and necrotizing enteroco-litis.

Coronaviruses are approximately 80 to 150 nm in diame-ter. They are seen as rounded or pleomorphic particles thathave club-shaped projections on their surface, called peplo-mers (296). These projections on the rounded particles givethe appearance of the corona of the sun, hence, the virions'name.

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Mortensen et al. (260) found that CVLP in stools in infantswith diarrhea were morphologically distinct from respiratorycoronaviruses in that the fecal CVLP had a flexible-ap-pearing fringe whereas the respiratory coronaviruses had amore rigid-appearing fringe. The CVLP in stools also hadmore closely spaced peplomers.Two respiratory coronaviruses, OC43 and 229E, are re-

sponsible for common colds and have been well character-ized. CVLP from stools have not been well characterized,since most investigators have not been able to cultivatethem. However, Gerna et al. (11, 122, 123) purified CVLPdirectly from the stools of two patients with gastroenteritis,using sucrose density gradient ultracentrifugation. Thisgroup also used the purified particles to study biophysicalcharacteristics. The two strains of purified human entericcoronavirus (HEC) of Gerna et al., HECV-24 and HECV-35,had a buoyant density of 1.20 g/cm3, a value in agreementwith those reported for human and animal coronaviruses.

In several epidemiologic studies, CVLP have been ob-served in the stools of normal individuals as well as indiarrheal or necrotizing enterocolitis stools (334), so that therole of CVLP in gastrointestinal disease has been ques-tioned. However, in some studies, a positive correlation hasbeen made between the incidence of fecal CVLP and gas-trointestinal illness. In a prospective study, Gerna et al. (122,123) found that, by EM, 16% of 208 gastroenteritis patientshad CVLP in their stools compared with only 2% of 182controls. Similarly, Mortensen et al. (248) found that 39% (49of 126) of diarrheal stools were positive for CVLP by EM. Inaddition to the correlation or lack of correlation of CVLPwith disease, some controversy or questions have arisenover the nature of the CVLPs. This is due in part to theinability of many investigators to propagate CVLP in vitro.In addition, in some instances the CVLP seen may not havebeen true virus particles, but rather, artifactual (283, 298,322). In some instances, the CVLP may be normal intestinalepithelial cell components, consisting of R-bodies (rod-containing bodies) and C-bodies (coccoid or glycocalycealbodies), which resemble CVLP (283). The possibility hasalso been considered that these CVLP may be mycoplasmas.However, efforts to propagate them on mycoplasma mediahave failed (260).

Viruses from human diarrheal stools have also been ob-served (by EM) that resemble coronaviruses, but are mor-phologically more similar to the newly described toroviruses(122). The human toro-like viruses are antigenically similarto the Breda virus. The toroviruses include Breda virusisolated from calves with diarrhea and Berne virus, whichwas isolated from a horse. The toroviruses are morphologi-cally similar to coronaviruses in that they are medium-sizedviruses with a peplomer-bearing envelope. However, theirpolypeptides are of different molecular weights than those ofcoronaviruses, and they appear to be negative-strandedRNA viruses, whereas the coronaviruses are positivestranded. Thus, in humans, some CVLP seen by EM mayactually be members of the torovirus group (122). In sum-mary, some investigators believe that the CVLP are aheterogeneous group of particles, some of which are truecoronaviruses.

Enteric coronavirus infections, or infections by CVLP,tend to occur in the cooler, drier months of the years. Insouthern Arizona, where a large percentage of gastroenter-itis appears to be caused by coronavirus, most of the casesoccur in the fall and early winter (260). Most cases ofgastroenteritis, as well as necrotizing enterocolitis, occur ininfants. Mortensen et al. (260) found that 49 patients with

CVLP-associated gastroenteritis ranged in age from 1 monthto 12 years. Of these 49 patients, 88%o were <2 years old and71% were <1 year old. The median duration of the illnesswas 7 days. Diarrhea occurred in 94%; vomiting, in 51%;fever, in 63%; and occult blood, in the stools of 18%.Eighteen percent had at least one other identifiable entericpathogen, including Salmonella, Shigella, and Campylobac-ter spp. and one enterovirus. Mortensen et al. (260) sug-gested that nursing might have a protective effect on pre-venting illness due to coronavirus-like agents, since weanedinfants seemed to be at risk for severe CVLP-associateddisease.

Serologic studies have been carried out for various pur-poses: (i) for epidemiologic purposes, (ii) to study theimmune response to enteric coronavirus infection, and (iii)to study possible cross-reactivity of fecal coronaviruses withknown respiratory coronaviruses.

In a retrospective study, Gerna et al. (122, 123) found that27% of sera from 62 patients with gastroenteritis reactedwith coronavirus OC43 by neutralization and HAI tests,whereas only 2% of control sera reacted. They also immu-nized guinea pigs and mice with the CVLP purified from thestools of two gastroenteritis patients by sucrose densitygradient ultracentrifugation. In an IEM test, they used theanimal sera to the two patients' strains, animal sera to OC43,and one patient's convalescent serum. In this test, theyfound a two-way cross between OC43, and their two humanenteric coronavirus strains. Thus, their fecal isolates ap-peared to share a common antigen(s) with respiratory coro-naviruses. However, Mortensen et al. (260) found that theirCVLP in stools did not cross-react with respiratory corona-virus OC43 or 229E by immunodiffusion or by IEM. Thus,there may be different antigenic forms of CVLP.The laboratory diagnosis of enteric coronavirus infection

or infection with CVLP is carried out primarily by directEM, since other laboratory tests are generally not available.Most investigators have not been able to propagate entericcoronaviruses, and no one has been able to propagate themin routine cell types. Mortensen et al. (260) could notpropagate CVLP from stools in (i) a number of routine celltypes, (ii) cell lines of human embryonic intestine or humanrectal tumor, or (iii) four strains of human embyronic tonsilfibroblasts. In some of the special cell types, five blindpasses were made, with negative results:

Coronavirus vaccines have not been developed for severalreasons. One, the role of enteric coronavirus in diseasecausation has not been firmly established in different geo-graphic areas of the world. Two, where coronavirus infec-tion may play a role, it is usually of minor importance,except in the southwest United States. Three, enteric coro-naviruses and CVLP are difficult to propagate in vitro. Four,long-term immunity to coronavirus infection has not beenstudied. Only short-term studies on convalescent sera havebeen carried out.Treatment consists of maintaining fluid and electrolyte

balance in infected infants, similar to that used for rotavirusand other serious viral gastroenteritis, when dehydration andelectrolyte imbalance can occur.

ACKNOWLEDGMENTSI thank Cynthia Howard and Joseph Zientarski for providing the

excellent electron photomicrographs and Donna Tieken for excel-lent editorial assistance.

LITERATURE CITED1. Agliano, A. M., A. Rossi, and A. Sanna. 1985. Isolation from

faecal specimens of new strains of human rotavirus primarily



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cytopathic for stationary cell culture without trypsin. Arch.Virol. 84:119-127.

2. Agus, S. G., R. Dolin, R. G. Wyatt, A. J. Tousimis, and R. S.Northrup. 1973. Acute infectious nonbacterial gastroenteritis:intestinal histopathology. Histologic and enzymatic alterationsduring illness produced by the Norwalk agent in man. Ann.Intern. Med. 79:18-25.

3. Albert, M. J., Y. Soenarto, and R. F. Bishop. 1982. Epidemi-ology of rotavirus diarrhea in Yogyakarta, Indonesia, as re-vealed by electrophoresis of genome RNA. J. Clin. Microbiol.16:731-733.

4. Albert, M. J., L. E. Unicomb, and R. F. Bishop. 1987. Culti-vation and characterization of human rotaviruses with "supershort" RNA patterns. J. Clin. Microbiol. 25:183-185.

5. Anderson, E. L., R. B. Belshe, J. Bartram, F. Crookshanks-Newman, R. M. Chanock, and A. Z. Kapikian. 1986. Evalua-tion of rhesus rotavirus vaccine (MMU 18006) in infants andyoung children. J. Infect. Dis. 153:823-831.

6. Appleton, H., M. Buckley, B. T. Thom, J. L. Cotton, and S.Henderson. 1977. Virus-like particles in winter vomiting dis-ease. Lancet i:409-411.

7. Ashley, C. R., E. 0. Caul, and W. K. Paver. 1978. Astrovirus-associated gastroenteritis in children. J. Clin. Pathol. 31:939-943.

8. Barnes, G. L., L. W. Doyle, P. H. Hewson, A. M. L. Knoches,J. A. McLellan, W. H. Kitchen, and R. F. Bishop. 1982. Arandomised trial of oral gammaglobulin in low-birth-weightinfants infected with rotavirus. Lancet i:1371-1373.

9. Barron-Romero, B. L., J. Barreda-Gonzalez, R. Doval-Ugalde,J. Zermeno-Equia Liz, and M. Huerta-Pena. 1985. Asympto-matic rotavirus infection in day care centers. J. Clin. Micro-biol. 22:116-118.

10. Bastardo, J. W., J. L. McKimm-Breschkin, S. Sonza, L. D.Mercer, and I. H. Holmes. 1981. Preparation and characteriza-tion of antisera to electrophoretically purified SA11 viruspolypeptides. Infect. Immun. 34:641-647.

11. Battaglia, M., N. Passarani, A. DiMatteo, and G. Gerna. 1987.Human enteric coronaviruses: further characterization andimmunoblotting of viral proteins. J. Infect. Dis. 155:140-143.

12. Beards, G. M., A. D. Campbell, N. R. Cottrell, J. S. M. Peiris,N. Rees, R. C. Sanders, J. A. Shirley, H. C. Wood, and T. H.Flewett. 1984. Enzyme-linked immunosorbent assays based onpolyclonal and monoclonal antibodies for rotavirus detection.J. Clin. Microbiol. 19:248-254.

13. Bellamy, K., P. S. Gardner, M. H. Hambling, S. Rice, and A. F.Bradburne. 1983. Enzyme-linked immunosorbent assay for thedetection of human rotavirus in stools. J. Virol. Methods 7:65-72.

14. Birch, C. J., N. I. Lehmann, A. J. Hawker, J. A. Marshall, andI. D. Gust. 1979. Comparison of electron microscopy, enzyme-linked immunosorbent assay, solid-phase radioimmunoassay,and indirect immunofluorescence for detection of human rota-virus antigen in faeces. J. Clin. Pathol. 32:700-705.

15. Birch, C. J., F. A. Lewis, M. L. Kennett, M. Homola, H.Pritchard; and I. D. Gust. 1977. A study of the prevalence ofrotavirus infection in children with gastroenteritis admitted toan infectious disease hospital. J. Med. Virol. 1:69-77.

16. Birch, C. J., S. M. Rodger, J. A. Marshall, and 1. D. Gust.1983. Replication of human rotavirus in cell culture. J. Med.Virol. 11:241-250.

17. Bishop, R. F., G. L. Barnes, E. Cipriani, and J. S. Lund. 1983.Clinical immunity after neonatal rotavirus infection. A pro-spective longitudinal study in young children. N. Engl. J. Med.309:72-76.

18. Bishop, R. F., G. P. Davidson, I. H. Holmes, and B. J. Ruck.1973. Virus particles in epithelial cells of duodenal mucosafrom children with acute non-bacterial gastroenteritis. Lancetii:1281-1283.

19. Bishop, R. F., G. P. Davidson, I. H. Holmes, and B. J. Ruck.1974. Detection of a new virus by electron microscopy offaecal extracts from children with acute gastroenteritis. Lanceti:149-151.

20. Black, R. E., M. H. Merson, A. S. M. M. Rahman, M. Yunis,

A. R. M. A. Alim, I. Huq, R. H. Yolken, and G. T. Curlin. 1980.A two-year study of bacterial, viral and parasitic agentsassociated with diarrhea in rural Bangladesh. J. Infect. Dis.142:660-664.

21. Blacklow, N. R., G. Cukor, M. K. Bedigian, P. Echeverria,H. B. Greenberg, D. S. Schreiber, and J. S. Trier. 1979.Immune response and prevalence of antibody to Norwalkenteritis virus as determined by radioimmunoassay. J. Clin.Microbiol. 10:903-909.

22. Blacklow, N. R., R. Dolin, D. S. Fedson, H. DuPont, R. S.Northrup, R. B. Hornick, and R. M. Chanock. 1972. Acuteinfectious nonbacterial gastroenteritis: etiology and pathogen-esis. Ann. Intern. Med. 76:933-1008.

23. Blacklow, N. R., P. Echeverria, and D. H. Smith. 1976. Sero-logic studies with reovirus-like enteritis agent. Infect. Immun.13:1563-1566.

24. Bohl, E. H., L. J. Saif, K. W. Theil, A. G. Agnes, and R. F.Cross. 1982. Porcine pararotaviruses: detection, differentiationfrom rotavirus, and pathogenesis in gnotobiotic pigs. J. Clin.Microbiol. 51:312-319.

25. Both, G. W., L. J. Seigman, A. R. Bellamy, N. Ikegami, A. J.Shatkin, and Y. Furuichi. 1984. Comparative sequence analysisof rotavirus genomic segment 6, the gene specifying viralsubgroups 1 and 2. J. Virol. 51:97-101.

26. Bradburne, A. F., J. D. Almeida, P. S. Gardner, R. B. Moosai,A. A. Nash, and R. R. A. Coombs. 1979. A solid-phase system(SPACE) for the detection and quantification of rotavirus infaeces. J. Gen. Virol. 44:615-623.

27. Brandt, C. D., C. W. Arndt, G. L. Evans, H. W. Kim, E. P.Staalings, W. J. Rodriguez, and R. H. Parrott. 1987. Evaluationof a latex test for rotavirus detection. J. Clin. Microbiol. 25:1800-1802.

28. Brandt, C. D., H. W. Kim, W. J. Rodriguez, J. 0. Arrobio,B. C. Jeffries, and R. H. Parrott. 1982. Rotavirus gastroente-ritis and weather. J. Clin. Microbiol. 16:478-482.

29. Brandt, C. D., H. W. Kim, W. J. Rodriguez, J. 0. Arrobio,B. C. Jeffries, and R. H. Parrott. 1986. Simultaneous infectionswith different enteric and respiratory tract viruses. J. Clin.Microbiol. 23:177-179.

30. Brandt, C. D., H. W. Kim, W. J. Rodriguez, J. 0. Arrobio,B. C. Jeffries, E. P. Stallings, C. Lewis, A. J. Miles, R. M.Chanock, A. Z. Kapikian, and R. H. Parrott. 1983. Pediatricviral gastroenteritis during eight years of study. J. Clin. Micro-biol. 18:71-78.

31. Brandt, C. D., H. W. Kim, W. J. Rodriguez, J. 0. Arrobio,B. C. Jeffries, E. P. Stallings, C. Lewis, A. J. Miles, M. K.Gardner, and R. H. Parrott. 1985. Adenovirus and pediatricgastroenteritis. J. Infect. Dis. 151:437-443.

32. Brandt, C. D., H. W. Kim, W. J. Rodriguez, L. Thomas, R. H.Yolken, J. 0. Arrobio, A. Z. Kapikian, R. H. Parrott, andR. M. Chanock. 1981. Comparison of direct electron micros-copy, immune electron microscopy, and rotavirus enzyme-linked immunosorbent assay for detection of gastroenteritisvirus in children. J. Clin. Microbiol. 13:976-981.

33. Brandt, C. D., H. W. Kim, R. H. Yolken, A. Z. Kapikian, J. 0.Arrobio, W. J. Rodriguez, R. G. Wyatt, R. M. Chanock, andR. H. Parrott. 1979. Comparative epidemiology of two rota-virus serotypes and other viral agents associated with pediatricgastroenteritis. Am. J. Epidemiol. 110:243-254.

34. Bridger, J. C., S. Pedley, and M. A. McCrae. 1986. Group Crotaviruses in humans. J. Clin. Microbiol. 23:760-763.

35. Brown, M. 1985. Selection of nonfastidious adenovirus speciesin 293 cells inoculated with stool specimens containing adeno-virus 40. J. Clin. Microbiol. 22:205-209.

36. Brown, M., M. Petric, and P. J. Middleton. 1984. Diagnosis offastidious enteric adenoviruses 40 and 41 in stool specimens. J.Clin. Microbiol. 20:334-338.

37. Brown, S. E., III, K. T. Sauer, M. Nations-Shields, D. S.Shields, J. G. Araujo, and R. L. Guerrant. 1982. Comparison ofpaired whole milk and dried filter paper samples for anti-enterotoxin and anti-rotavirus activities. J. Clin. Microbiol. 16:103-106.

38. Brussow, H., H. Hilpert, I. Walther, J. Sidoti, C. Mietens, and

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P. Bachman. 1987. Bovine milk immunoglobulins for passiveimmunity to infantile rotavirus gastroenteritis. J. Clin. Micro-biol. 25:982-986.

39. Bryden, A. S., H. A. Davies, R. E. Hadley, T. H. Flewett, C. A.Morris, and P. Oliver. 1975. Rotavirus enteritis in the WestMidlands during 1974. Lancet ii:241-243.

40. Cameron, D. J. S., R. F. Bishop, A. A. Veenstra, and G. L.Barnes. 1978. Noncultivable viruses and neonatal diarrhea:fifteen-month survey in a newborn special care nursery. J.Clin. Microbiol. 8:93-98.

41. Carlson, J. A. K., P. J. Middleton, M. T. Szymanski, J. Huber,and M. Petric. 1978. Fatal rotavirus gastroenteritis. An analy-sis of 21 cases. Am J. Dis. Child. 132:477-479.

42. Caul, E. O., and H. Appleton. 1982. The electron microscopaland physical characteristics of small round human fecal vi-ruses: an interim scheme for classification. J. Med. Virol. 9:257-265.

43. Cevenini, R., F. Rumpianesi, R. Mazzaracchio, M. Donati, E.Falcieri, and R. Lazzari. 1983. Evaluation of a new latexagglutination test for detecting human rotavirus in faeces. J.Infect. 7:130-133.

44. Cevenini, R., F. Rumpianesi, R. Mazzaracchio, M. Donati, E.Falcieri, and I. Sarov. 1984. A simple immunoperoxidasemethod for detecting enteric adenovirus and rotavirus in cellculture. J. Infect. 8:22-27.

45. Champsaur, H., M. Henry-Amar, D. Goldszmidt, J. Prevot, M.Bourjouane, E. Questiaux, and C. Bach. 1984. Rotavirus car-riage, asymptomatic infection, and disease in the first twoyears of life. II. Serological response. J. Infect. Dis. 149:675-682.

46. Champsaur, H., E. Questiaux, J. Prevot, M. Henry-Amar, D.Goldszmidt, M. Bourjouane, and C. Bach. 1984. Rotaviruscarriage, asymptomatic infection, and disease in the first twoyears of life. I. Virus shedding. J. Infect. Dis. 149:667-674.

47. Chanock, S. J., E. A. Wenske, and B. N. Fields. 1983. Humanrotaviruses and genome RNA. J. Infect. Dis. 148:49-50.

48. Chen, G.-M., T. Hung, J. C. Bridger, and M. A. McCrae. 1985.Chinese adult rotavirus is a Group B rotavirus. Lancet ii:1123-1124.

49. Chernesky, M., S. Castriciano, J. Mahony, and D. DeLong.1985. Examination of the Rotazyme II enzyme immunoassayfor the diagnosis of rotavirus gastroenteritis. J. Clin. Micro-biol. 22:462-464.

50. Cheung, E. Y., S. I. Hnatko, H. Gunning, and J. Wilson. 1982.Comparison of Rotazyme and direct electron microscopy fordetection of rotavirus in human stools. J. Clin. Microbiol. 16:562-563.

51. Chiba, S., I. Nakamura, S. Urasawa, S. Nakata, K. Taniguchi,K. Fujinaga, and T. Nakao. 1983. Outbreak of infantile gas-troenteritis due to type 40 adenovirus. Lancet ii:954-957.

52. Chiba, S., S. Nakata, T. Urasawa, S. Urasawa, T. Yokoyama,Y. Morita, K. Taniguchi, and T. Nakao. 1986. Protective effectof naturally acquired homotypic and heterotypic rotavirusantibodies. Lancet ii:417-421.

53. 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.

54. Chiba, S., Y. Sakuma, R. Kogasaka, M. Akihara, H. Te-rashima, K. Horino, and T. Nakao. 1980. Fecal shedding ofvirus in relation to the days of illness in infantile gastroenteritisdue to calicivirus. J. Infect. Dis. 142:247-249.

55. Christy, C., H. P. Madore, J. J. Treanor, K. Pray, A. Z.Kapikian, R. M. Chanock, and R. Dolin. 1986. Safety andimmunogenicity of live attenuated rhesus monkey rotavirusvaccine. J. Infect. Dis. 154:1045-1047.

56. Chrystie, I. L., B. Totterdell, M. J. Baker, J. W. Scopes, andJ. E. Banatvala. 1975. Rotavirus infections in a maternity unit.Lancet ii:79.

57. Chrystie, I. L., B. M. Totterdell, and J. E. Banatvala. 1978.Asymptomatic endemic rotavirus infections in the newborn.Lancet i:1176-1178.

58. Chrystie, I. L., B. M. Totterdell, and J. E. Banatvala. 1983.

False positive Rotazyme tests on faecal samples from babies.Lancet ii:1028.

59. Clark, H. F., K. T. Dolan, P. Horton-Slight, J. Palmer, andS. A. Plotkin. 1985. Diverse serologic response to rotavirusinfection of infants in a single epidemic. Pediatr. Infect. Dis. 4:626-631.

60. Clark, H. F., T. Furukawa, L. M. Bell, P. A. Offit, P. A.Perrella, and S. A. Plotkin. 1986. Immune response of infantsand children to low-passage bovine rotavirus (strain WC3).Am. J. Dis. Child. 140:350-356.

61. Clark, H. F., Y. Hoshino, L. M. Bell, J. Groff, G. Hess, P.Bachman, and P. A. Offit. 1987. Rotavirus isolate W161 repre-senting a presumptive new human serotype. J. Clin. Microbiol.25:1757-1762.

62. Cook, D. A., A. Zbitnew, G. Dempster, and J. W. Gerrard.1978. Detection of antibody to rotavirus by counterimmunoe-lectrophoresis in human serum, colostrum, and milk. J. Pe-diatr. 93:967-970.

63. Coulson, B. S., and I. H. Holmes. 1984. An improved enzyme-linked immunosorbent assay for the detection of rotavirus infaeces of neonates. J. Virol. Methods 8:165-179.

64. Coulson, B. S., L. E. Unicomb, G. A. Pitson, and R. F. Bishop.1987. Simple and specific enzyme immunoassay using mono-clonal antibodies for serotyping human rotaviruses. J. Clin.Microbiol. 25:509-515.

65. Cruickshank, J. G., J. H. M. Axton, and 0. F. Webster. 1974.Viruses in gastroenteritis. Lancet i:1353.

66. Cubitt, W. D. 1982. Rotavirus infection: an unexpected hazardin units caring for the elderly. Geriat. Med. Today 1:33-38.

67. Cubitt, W. D., and D. A. McSwiggan. 1981. Calicivirus gas-troenteritis in North West London. Lancet ii:975-977.

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

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

70. 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 the elderly. J. Clin. Pathol. 34:924-926.

71. Cukor, G., N. R. Blacklow, F. E. Capozza, Z. F. K. Panjvani,and F. Bednarek. 1979. Persistence of antibodies to rotavirus inhuman milk. J. Clin. Microbiol. 9:93-96.

72. Cukor, G., N. R. Blacklow, P. Echeverria, M. K. Bedigian, H.Puruggan, and V. Basaca-Sevilla. 1980. Comparative study ofthe acquisition of antibody to Norwalk virus in pediatricpopulations. Infect. Immun. 29:822-823.

73. Dai, G.-Z., M.-S. Sun, S.-Q. Liu, X.-F. Ding, T.-D. Chen, L.-C.Wang, D.-P. Du, G. Zhao, Y. Su, J. Li, W.-M. Xu, T.-H. Li,and X.-X Chen. 1987. First report of an epidemic of diarrhoeain human neonates involving the new rotavirus and biologicalcharacteristics of the epidemic virus strain (KMB/R85). J.Med. Virol. 22:365-373.

74. Davidson, G. P., and G. L. Barnes. 1979. Structural andfunctional abnormalities of the small intestine in infants andyoung children with rotavirus enteritis. Acta Paediatr. Scand.68:181-186.

75. Davidson, G. P., R. J. Hogg, and C. P. Kirubakaran. 1983.Serum and intestinal immune response to rotavirus enteritis inchildren. Infect. Immun. 40:447-452.

76. Davidson, G. P., R. R. W. Townley, R. F. Bishop, I. H. Holmes,and B. J. Ruck. 1975. Importance of a new virus in acutesporadic enteritis in children. Lancet i:242-246.

77. de Jong, J. C., R. Wigand, A. H. Kidd, G. Wadell, J. G.Kapsenberg, C. J. Muzerie, A. G. Wermenbol, and R.-G.Firtzlaff. 1983. Candidate adenoviruses 40 and 41: fastidiousadenoviruses from human infant stool. J. Med. Virol. 11:215-231.

78. Dennehy, P. H., and G. Peter. 1985. Risk factors associatedwith nosocomial rotavirus infection. Am. J. Dis. Child. 139:935-939.

79. DeSilva, L. M., M. Hanlon, and J. Stanton. 1983. Rotavirus



r.asm.org/

Dow

nloaded from

http://cmr.asm.org/


infection. Med. J. Aust. 2:602-603.80. de Torres, B. V., R. M. de lUa, and J. Esparza. 1978. Epide-

miological aspects of rotavirus infection in hospitalized Vene-zuelan children with gastroenteritis. Am. J. Trop. Med. Hyg.27:567-572.

81. Dimitrov, D. H., M. K. Estes, S. M. Rangelova, L. M. Shinda-rov, J. L. Melnick, and D. Y. Graham. 1983. Detection ofantigenically distinct rotaviruses from infants. Infect. Immun.41:523-526.

82. Dimitrov, D. H., D. Y. Graham, and M. K. Estes. 1985.Detection of rotaviruses by nucleic acid hybridization withcloned DNA of simian rotavirus SA11 genes. J. Infect. Dis.152:293-300.

83. Doern, G. V., J. E. Herrmann, P. Henderson, D. Stobbs-Walro,D. M. Perron, and N. R. Blacklow. 1986. Detection of rotaviruswith a new polyclonal antibody enzyme immunoassay (Ro-tazyme II) and a commercial latex agglutination tests (Ro-talex): comparison with a monoclonal antibody enzyme immu-noassay. J. Clin. Microbiol. 23:226-229.

84. Dolin, R., N. R. Blacklow, H. DuPont, R. F. Buscho, R. G.Wyatt, J. A. Kasel, R. Hornick, and R. M. Chanock. 1972.Biological properties of Norwalk agent of acute infectiousnonbacterial gastroenteritis. Proc. Soc. Exp. Biol. Med. 140:578-583.

85. Dolin, R., N. R. Blacklow, H. DuPont, S. Formal, R. F. Buscho,J. A. Kasel, R. P. Chames, R. Hornick, and R. M. Chanock.1971. Transmission of acute infectious nonbacterial gastroen-teritis to volunteers by oral administration of stool filtrates. J.Infect. Dis. 123:307-312.

86. Dolin, R., A. G. Levy, R. G. Wyatt, T. S. Thornhill, and J. D.Gardner. 1975. Viral gastroenteritis induced by the Hawaiiagent. Jejunal histopathology and serologic response. Am. J.Med. 59:761-768.

87. Dolin, R., R. C. Reichman, K. D. Roessner, T. S. Tralka, R. T.Schooley, W. Gary, and D. Morens. 1982. Detection by immuneelectron microscopy of the Snow Mountain agent of acute viralgastroenteritis. J. Infect. Dis. 146:184-189.

88. Dolin, R., K. D. Roessner, J. J. Treanor, R. C. Reichman, M.Phillips, and H. P. Madore. 1986. Radioimmunoassay fordetection of the Snow Mountain agent of viral gastroenteritis.J. Med. Virol. 19:11-18.

89. Dowling, J. M., and H. Wynne. 1981. Role of enteric adenovi-ruses and rotaviruses in infantile gastroenteritis. Lancet ii:305-306.

90. Duffy, L. C., T. E. Byers, M. Riepenhoff-Talty, L. J. La Scolea,M. Zielezny, and P. L. Ogra. 1986. The effects of infant feedingon rotavirus-induced gastroenteritis: a prospective study. Am.J. Public Health 76:259-263.

91. DuPont, H. L. 1984. Rotaviral gastroenteritis-some recentdevelopments. J. Infect. Dis. 149:663-666.

92. Dyall-Smith, M. L., A. A. Azad, and I. H. Holmes. 1983. Genemapping of rotavirus double-stranded RNA segments byNorthern blot hybridization: application to segments 7, 8, and9. J. Virol. 46:317-320.

93. Dyall-Smith, M. L., and I. H. Holmes. 1981. Comparisons ofrotavirus polypeptides by limited proteolysis: close similarityof certain polypeptides of different strains. J. Virol. 40:720-728.

94. Dyall-Smith, M. L., and I. H. Holmes. 1981. Gene-codingassignments of rotavirus double-stranded RNA segments 10and 11. J. Virol. 38:1099-1103.

95. Dyall-Smith, M. L., and I. H. Holmes. 1984. Sequence homol-ogy between human and animal rotavirus serotype-specificglycoproteins. Nucleic Acids Res. 12:3973-3982.

96. Ebina, T., A. Sato, K. Umezu, N. Ishida, S. Ohyama, A.Oizumi, K. Aikawa, S. Katagiri, N. Katsushima, A. Imai, S.Kitaoka, H. Suzuki, and T. Konno. 1985. Prevention of rota-virus infection by oral administration of cow colostrum con-taining anti-human rotavirus antibody. Med. Microbiol. Immu-nol. 174:177-185.

97. Ebina, T. A., K. Umezu, S. Ohyama, K. Aikawa, N. Kat-sushima, S. Kitaoka, A. Sato, N. Ishida, A. Ohizumi, S.Katagiri, A. Imai, H. Suzuki, and T. Konno. 1983. Prevention

of rotavirus infection by cow colostrum containing antibodyagainst human rotavirus. Lancet ii:1029-1030.

98. Echeverria, P., N. R. Blacklow, G. G. Cukor, S. Vibulbandhit-kit, S. Changchawalit, and P. Boonthai. 1983. Rotavirus as acause of severe gastroenteritis in adults. J. Clin. Microbiol. 18:663-667.

99. Eiden, J., H. M. Lederman, S. Vonderfecht, and R. Yolken.1986. T-cell-deficient mice display normal recovery from ex-perimental rotavirus infection. J. Virol. 57:706-708.

100. Eiden, J., S. Sato, and R. Yolken. 1987. Specificity of dothybridization assay in the presence of rRNA for detection ofrotaviruses in clinical specimens. J. Clin. Microbiol. 25:1809-1811.

101. Elden, J., S. Vonderfecht, K. Theil, A. Torres-Medina, andR. H. Yolken. 1986. Genetic and antigenic relatedness ofhuman and animal strains of antigenically distinct rotaviruses.J. Infect. Dis. 154:972-981.

102. Engvall, E., and P. Perlmann. 1972. Enzyme-linked immuno-sorbent assay, ELISA. III. Quantitation of specific antibodiesby enzyme-labeled anti-immunoglobulin in antigen-coatedtubes. J. Immunol. 109:129-135.

103. Espejo, R. T., E. Calderon, N. Gonzalez, A. Salomon, A.Martuscelli, and P. Romero. 1979. Presence of two distincttypes of rotavirus in infants and young children hospitalizedwith acute gastroenteritis in Mexico City, 1977. J. Infect. Dis.139:474-477.

104. Espejo, R. T., 0. Munoz, F. Serafin, and P. Romero. 1980. Shiftin the prevalent human rotavirus detected by ribonucleic acidsegment differences. Infect. Immun. 27:351-354.

105. Espejo, R. T., F. Puerto, C. Soler, and N. Gonzalez. 1984.Characterization of human pararotavirus. Infect. Immun. 44:112-116.

106. Estes, M. K., E. L. Palmer, and J. Obijeski. 1983. Rotaviruses:a review. Curr. Top. Microbiol. Immunol. 105:123-184.

107. Fernbach, S. K., and J. D. Lloyd-Still. 1984. The radiographicfindings in severe rotavirus-induced colitis. J. Can. Assoc.Radiol. 35:192-194.

108. Flewett, T. H. 1976. Diagnosis of enteritis virus. Proc. R. Soc.Med. 69:693-696.

109. Flewett, T. H., A. S. Bryden, and H. Davies. 1973. Virusparticles in gastroenteritis. Lancet ii:1497.

110. Flewett, T. H., A. S. Bryden, H. Davies, and C. A. Morris.1975. Epidemic viral enteritis in a long-stay children's ward.Lancet i:4-5.

111. Flewett, T. H., A. S. Bryden, H. Davies, G. N. Woode, J. C.Bridger, and J. M. Derrick. 1974. Relation between virusesfrom acute gastroenteritis of children and newborn calves.Lancet ii:61-63.

112. Flewett, T. H., and H. Davies. 1976. Calicivirus in man. Lanceti:311.

113. Flewett, T. H., and G. N. Woode. 1978. The rotaviruses. Arch.Virol. 57:1-25.

114. Flores, J., H. B. Greenberg, H. Myslinski, A. R. Kalica, R. G.Wyatt, A. Z. Kapikian, and R. M. Chanock. 1982. Use oftranscription probes for genotyping rotavirus reassortants.Virology 121:288-295.

115. Flores, J., I. Perez, L. White, M. Perez, A. R. Kalica, R.Marquina, R. G. Wyatt, A. Z. Kapikian and R. M. Chanock.1982. Genetic relatedness among human rotaviruses as deter-mined by RNA hybridization. Infect. Immun. 37:648-655.

116. Flores, J., R. H. Purcell, I. Perez, R. G. Wyatt, E. Boeggeman,M. Sereno, L. White, R. M. Chanock, and A. Z. Kapikian.1983. A dot hybridization assay for detection of rotavirus.Lancet i:555-559.

117. Fox, J. P., C. D. Hall, and M. C. Cooney. 1977. The Seattlevirus watch. VII. Observations on adenovirus infections. Am.J. Epidemiol. 105:362-386.

118. Fragoso, M., A. Kumar, and D. L. Murray. 1986. Rotavirus innasopharyngeal secretions of children with upper respiratorytract infections. Diagn. Microbiol. Infect. Dis. 4:87-88.

119. Gary, G. W., Jr., J. C. Hierholzer, and R. E. Black. 1979.Characteristics of noncultivable adenoviruses associated withdiarrhea in infants: a new subgroup of human adenoviruses. J.

VOL. 2, 1989


r.asm.org/

Dow

nloaded from

http://cmr.asm.org/

82 CHRISTENSEN

Clin. Microbiol. 10:96-103.120. Gary, G. W., Jr., J. E. Kaplan, S. E. Stine, and L. J. Anderson.

1985. Detection of Norwalk virus antibodies and antigen with abiotin-avidin immunoassay. J. Clin. Microbiol. 22:274-278.

121. Gerna, G., N. Passarani, M. Battaglia, and E. Percivalle. 1984.Rapid serotyping of human rotavirus strains by solid-phaseimmune electron microscopy. J. Clin. Microbiol. 19:273-278.

122. Gerna, G., N. Passarani, M. Battaglia, and E. G. Rondanelli.1985. Human enteric coronaviruses: antigenic relatedness tohuman coronavirus OC43 and possible etiologic role in viralgastroenteritis. J. Infect. Dis. 151:796-803.

123. Gerna, G., N. Passarani, P. M. Cereda, and M. Battaglia. 1984.Antigenic relatedness of human enteric coronavirus strains tohuman coronavirus OC43: a preliminary report. J. Infect. Dis.150:618-619.

124. Gerna, G., N. Passarani, A. Sarasini, and M. Battaglia. 1985.Characterization of serotypes of human rotavirus strains bysolid-phase immune electron microscopy. J. Infect. Dis. 152:1143-1151.

125. Gerna, G., A. Sarasini, N. Passarani, M. Torsellini, M. Parea,and M. Battaglia. 1987. Comparative evaluation of a commer-cial enzyme-linked immunoassay and solid-phase immuneelectron microscopy for rotavirus detection in stool specimens.J. Clin. Microbiol. 25:1137-1139.

126. Giaquinto, C., G. Errico, E. Ruga, I. Naso, and R. D'Elia. 1986.Evaluation of ELISA test for rotavirus diagnosis in neonates.J. Pediatr. 109:565-566.

127. Goldwater, P. N., I. L. Chrystie, and J. E. Banatvala. 1979.Rotaviruses and the respiratory tract. Br. Med. J. 2:1551.

128. Graham, F. L., J. Smiley, W. C. Russell, and R. Nairn. 1977.Characteristics of a human cell line transformed by DNA fromhuman adenovirus type 5. J. Gen. Virol. 36:59-72.

129. Grauballe, P. C., K. Hjelt, P. A. Krasilnikoff, and P. O. Schiotz.1981. Elisa for rotavirus-specific secretary IgA in human sera.Lancet ii:588-589.

130. Green, M. 1970. Oncogenic viruses. Annu. Rev. Biochem. 39:701-756.

131. Green, M., J. K. Mackey, W. S. M. Wold, and P. Rigden. 1979.Thirty-one human adenovirus serotypes (Adl-Ad3l) form fivegroups (A-E) based upon DNA genome homologies. Virology93:481-492.

132. Greenberg, H., V. McAuliffe, J. Valdesuso, R. Wyatt, J. Flores,A. Kalica, Y. Hoshino, and N. Singh. 1983. Serological analysisof the subgroup protein of rotavirus, using monoclonal anti-bodies. Infect. Immun. 39:91-99.

133. Greenberg, H. B., J. R. Valdesuso, A. R. Kalica, R. G. Wyatt,V. J. McAuliffe, A. Z. Kapikian, and R. M. Chanock. 1981.Proteins of Norwalk virus. J. Virol. 37:994-999.

134. Greenberg, H. B., J. R. Valdesuso, A. Z. Kapikian, R. M.Chanock, R. G. Wyatt, W. Szmaness, J. Larrick, J. Kaplan,R. H. Gilman, and D. A. Sack. 1979. Prevalence of antibody tothe Norwalk virus in various countries. Infect. Immun. 26:270-273.

135. Greenberg, H. B., J. Valdesuso, K. Van Wyke, K. Midthun, M.Walsh, V. McAuliffe, R. G. Wyatt, A. R. Kalica, J. Flores, andY. Hoshino. 1983. Production and preliminary characterizationof monoclonal antibodies directed at two surface proteins ofrhesus rotavirus. J. Virol. 47:267-275.

136. Greenberg, H. B., J. Valdesuso, R. H. Yolken, E. Gangarosa,W. Gary, R. G. Wyatt, T. Konno, H. Suzuki, R. M. Chanock,and A. Z. Kapikian. 1979. Role of Norwalk virus in outbreaksof nonbacterial gastroenteritis. J. Infect. Dis. 139:564-568.

137. Greenberg, H. B., R. G. Wyatt, A. R. Kalica, R. H. Yolken, R.Black, A. Z. Kapikian, and R. M. Chanock. 1981. New insightsin viral gastroenteritis. Perspect. Virol. 11:163-187.

138. Greenberg, H. B., R. G. Wyatt, J. Valdesuso, A. R. Kalica,W. T. London, R. M. Chanock, and A. Z. Kapikian. 1978.Solid-phase microtiter radioimmunoassay for detection of theNorwalk strain of acute nonbacterial, epidemic gastroenteritisvirus and its antibodies. J. Med. Virol. 2:97-108.

139. Grunow, J. E., S. F. Dunton, and J. L. Waner. 1985. Humanrotavirus-like particles in a hepatic abscess. J. Pediatr. 106:73-76.

140. Haikala, 0. J., J. 0. Kokkonen, M. K. Leinonen, T. Nurmi, R.Mantyjarvi, and H. K. Sarkkinen. 1983. Rapid detection ofrotavirus in stool by latex agglutination: comparison withradioimmunoassay and electron microscopy and clinical eval-uation of the test. J. Med. Virol. 11:91-97.

141. Halonen, P., H. Sarkkinen, P. Arstila, E. Hjertsson, and E.Torfason. 1980. Four-layer radioimmunoassay for detection ofadenovirus in stool. J. Clin. Microbiol. 11:614-617.

142. Halvorsrud, J., and I. Orstavik. 1980. An epidemic of rota-virus-associated gastroenteritis in nursing home for the elderly.Scand. J. Infect. Dis. 12:161-164.

143. Hammond, G., C. Hannan, T. Yeh, K. Fischer, G. Mauthe, andS. E. Straus. 1987. DNA hybridization for diagnosis of entericadenovirus infection from directly spotted fecal specimens. J.Clin. Microbiol. 25:1881-1885.

144. Hammond, G. W., G. S. Ahluwalia, F. G. Barker, G. Horsman,and P. R. Hazelton. 1982. Comparison of direct and indirectenzyme immunoassays with direct ultracentrifugation beforeelectron microscopy for detection of rotaviruses. J. Clin.Microbiol. 16:53-59.

145. Hammond, G. W., G. S. Ahluwalia, and P. R. Hazelton. 1984.Detection of human rotavirus in fecal specimens by a commer-cial latex-agglutination test. J. Infect. Dis. 149:1021.

146. Hasegawa, A., S. Matsuno, S. Inouye, R. Kono, Y. Tsurukubo,A. Mukoyama, and Y. Saito. 1982. Isolation of human rotavi-ruses in primary cultures of monkey kidney cells. J. Clin.Microbiol. 16:387-390.

147. Heimer, G. V., and W. D. Cubitt. 1983. Improved immunoflu-orescence techniques with microplates for the detection of Mand G immunoglobulins against rotavirus. J. Virol. Methods 6:31-39.

148. Herrmann, J. E., R. W. Hudson, N. R. Blacklow, and W. D.Cubitt. 1987. Marin County agent, an astrovirus. Lancet ii:743.

149. Herrmann, J. E., G. P. Kent, N. A. Nowak, J. Brondum, andN. R. Blacklow. 1986. Antigen detection in the diagnosis ofNorwalk virus gastroenteritis. J. Infect. Dis. 154:547-548.

150. Herrmann, J. E., N. A. Nowak, and N. R. Blacklow. 1985.Detection of Norwalk virus in stools by enzyme immunoassay.J. Med. Virol. 17:127-133.

151. Herrmann, J. E., D. M. Perron, N. R. Blacklow, G. Cukor,P. J. Krause, J. S. Hyams, H. J. Barrett, and P. L. Ogra. 1985.Enzyme immunoassay with monoclonal antibodies for thedetection of rotavirus in stool specimens. J. Infect. Dis. 152:830-832.

152. Hieber, J. P., S. Shelton, J. D. Nelson, J. Leon, and E. Mohs.1978. Comparison of human rotavirus disease in tropical andtemperate settings. Am. J. Dis. Child. 132:853-858.

153. Hierholzer, J. C., K. H. Johansson, L. J. Anderson, C. J. Tsou,and P. E. Halonen. 1987. Comparison of monoclonal time-resolved fluoroimmunoassay with monoclonal capture-biotiny-lated detector enzyme immunoassay for adenovirus antigendetection. J. Clin. Microbiol. 25:1662-1667.

154. Hilpert, H., H. Brussow, C. Mietens, J. Sidoti, L. Lerner, andH. Werchau. 1987. Use of bovine milk concentrate containingantibody to rotavirus to treat rotavirus gastroenteritis in in-fants. J. Infect. Dis. 156:158-166.

155. Hjelt, K., P. C. Grauballe, L. Andersen, P. 0. Schiotz, P.Howitz, and P. A. Krasilnikoff. 1986. Antibody response inserum and intestine in children up to six months after anaturally acquired rotavirus gastroenteritis. J. Pediatr. Gastro-enterol. Nutr. 5:74-80.

156. Hjelt, K., P. C. Grauballe, L. Henriksen, and P. A. Krasilni-koff. 1985. Rotavirus infections among the staff of a generalpaediatric department. Acta Paediatr. Scand. 74:617-618.

157. Hjelt, K., P. C. Grauballe, 0. H. Nielsen, P. 0. Schiotz, andP. A. Krasilnikoff. 1985. Rotavirus antibodies in the motherand her breast-fed infant. J. Pediatr. Gastroenterol. Nutr. 4:414-420.

158. Hjelt, K., P. C. Grauballe, A. Paerregaard, 0. H. Nielsen, andP. A. Krasilnikoff. 1987. Protective effect of preexisting rota-virus-specific immunoglobulin A against naturally acquiredrotavirus infection in children. J. Med. Virol. 21:39-47.

159. Hjelt, K., P. C. Grauballe, P. 0. Schiotz, L. Andersen, and



r.asm.org/

Dow

nloaded from

http://cmr.asm.org/


P. A. Krasilnikoff. 1985. Intestinal and serum immune re-

sponse to a naturally acquired rotavirus gastroenteritis inchildren. J. Pediatr. Gastroenterol. Nutr. 4:60-66.

160. Hjelt, K., P. A. Krasilnikoff, P. C. Grauballe, and S. W.Rasmussen. 1985. Nosocomial acute gastroenteritis in a pae-diatric department, with special reference to rotavirus infec-tions. Acta Paediatr. Scand. 74:89-95.

161. Hjelt, K., P. A. Krasilnikoff, P. C. Grauballe, and S. W.Rasmussen. 1985. Clinical features in hospitalized children withacute gastroenteritis. Acta Paediatr. Scand. 74:96-101.

162. Hogg, R. J., and G. P. Davidson. 1982. Improved specificity ofELISA for rotavirus. Aust. Paediatr. J. 18:184-185.

163. Holmes, I. H. 1983. Rotaviruses, p. 359-423. In W. K. Joklik(ed.), The Reoviridae. Plenum Publishing Corp., New York.

164. Holmes, I. H., M. Mathan, P. Bhat, M. J. Albert, S. P.Swaminathan, P. P. Maiya, S. M. Pereira, and S. J. Baker.1974. Orbiviruses and gastroenteritis. Lancet ii:658-659.

165. Hoshino, Y., M. M. Sereno, K. Midthun, J. Flores, A. Z.Kapikian, and R. M. Chanock. 1985. Independent segregationof two antigenic specificities (VP3 and VP7) involved inneutralization of rotavirus infectivity. Proc. Natl. Acad. Sci.USA 82:8701-8704.

166. Hoshino, Y., R. G. Wyatt, H. B. Greenberg, J. Flores, and A. Z.Kapikian. 1984. Serotypic similarity and diversity of rotavi-ruses of mammalian and avian origin as studied by plaque-reduction neutralization. J. Infect. Dis. 149:694-702.

167. Hovi, T., V. Vaisanen, P. Ukkonen, and C.-H. von Bonsdorff.1982. Solid-phase enzyme immunoassay for rotavirus antigen:faecal protease activity as a reason for false-negative results. J.Virol. Methods 5:45-53.

168. Huebner, R. J. 1967. Adenovirus-directed tumor and T anti-gens. Perspect. Virol. 5:147-166.

169. Hughes, J. H., A. V. Tuomari, D. R. Mann, and V. V.Hamparian. 1984. Latex immunoassay for rapid detection ofrotavirus. J. Clin. Microbiol. 20:441-447.

170. Hung, T., G. Chen, C. Wang, Z. Chou, T. Chao, W. Ye, H.Yao, and K. Meng. 1983. Rotavirus-like agent in adult non-

bacterial diarrhoea in China. Lancet ii:1078-1079.171. Hung, T., G. Chen, C. Wang, H. Yao, Z. Fang, T. Chao, Z.

Chou, W. Ye, X. Chang, S. Den, X. Liong, and W. Chang. 1984.Waterborne outbreak of rotavirus diarrhoea in adults in Chinacaused by a novel rotavirus. Lancet i:1139-1142.

172. Hyams, J. S., P. J. Krause, and P. A. Gleason. 1981. Lactosemalabsorption following rotavirus infection in young children.J. Pediatr. 99:916-918.

173. Inouye, S., S. Matsuno, and H. Yamaguchi. 1984. Efficientcoating of the solid phase with rotavirus antigens for enzyme-linked immunosorbent assay of immunoglobulin A antibody infeces. J. Clin. Microbiol. 19:259-263.

174. Jacobsson, P. A., M. E. Johansson, and G. Wadell. 1979.Identification of an enteric adenovirus by immunoelectroosmo-phoresis (EIOP) technique. J. Med. Virol. 3:307-312.

175. Johansson, M. E., I. Uhnoo, A. H. Kidd, C. R. Madeley, and G.Wadell. 1980. Direct identification of enteric adenovirus, acandidate new serotype, associated with infantile gastroenter-itis. J. Clin. Microbiol. 12:95-100.

176. Johansson, M. E., I. Uhnoo, L. Svensson, C.-A. Pettersson, andG. Wadell. 1985. Enzyme-linked immunosorbent assay fordetection of enteric adenovirus 41. J. Med. Virol. 17:19-27.

177. Joncas, J., A. Moisan, and V. Pavilanis. 1962. Incidence ofadenovirus infection: a family study. Can. Med. Assoc. J. 87:52-58.

178. Julkunen, I., J. Savolainen, A. Hautanen, and T. Hovi. 1985.Detection of rotavirus in faecal specimens by enzyme immu-noassay, latex agglutination and electron microscopy. Scand.J. Infect. Dis. 17:245-249.

179. Kalica, A. R., J. Flores, and H. B. Greenberg. 1983. Identifi-cation of the rotaviral gene that codes for hemagglutination andprotease-enhanced plaque formation. Virology 125:194-205.

180. Kalica, A. R., H. B. Greenberg, R. G. Wyatt, J. Flores, M. M.Sereno, A. Z. Kapikian, and R. M. Chanock. 1981. Genes ofhuman (strain Wa) and bovine (strain UK) rotaviruses thatcode for neutralization and subgroup antigens. Virology 112:

385-390.181. Kalica, A. R., R. H. Purcell, M. M. Sereno, R. G. Wyatt, H. W.

Kim, R. M. Chanock, and A. Z. Kapikian. 1977. A microtitersolid-phase radioimmunoassay for detection of human reo-virus-like agent in stools. J. Immunol. 118:1275-1279.

182. Kalica, A. R., M. M. Sereno, R. G. Wyatt, C. A. Mebus, R. M.Chanock, and A. Z. Kapikian. 1978. Comparison of human andanimal rotavirus strains by gel electrophoresis of viral RNA.Virology 87:247-255.

183. Kantharidis, P., M. L. Dyall-Smith, and I. H. Holmes. 1983.Completion of the gene-coding assignments of SA11 rotavirus:gene products of segments 7, 8, and 9. J. Virol. 48:330-334.

184. Kapikian, A. Z., J. Flores, Y. Hoshino, R. I. Glass, K. Midthun,M. Gorziglia, and R. M. Chanock. 1986. Rotavirus: the majoretiologic agent of severe infantile diarrhea may be controllableby a "Jennerian" approach to vaccination. J. Infect. Dis. 153:815-822.

185. Kapikian, A. Z., H. W. Kim, R. G. Wyatt, W. L. Cline, J. 0.Arrobio, C. D. Brandt, W. J. Rodriguez, D. A. Sack, R. M.Chanock, and R. H. Parrott. 1976. Human reovirus-like agentas the major pathogen associated with "winter" gastroenteritisin hospitalized infants and young children. N. Engl. J. Med.294:965-972.

186. Kapikian, A. Z., H. W. Kim, R. G. Wyatt, W. J. Rodriguez, S.Ross, W. L. Cline, R. H. Parrott, and R. M. Chanock. 1974.Reovirus-like agent in stools: association with infantile diar-rhea and development of serologic tests. Science 185:1049-1053.

187. Kapikian, A. Z., R. G. Wyatt, R. Dolin, T. S. Thornhill, A. R.Kalica, and R. M. Chanock. 1972. Visualization by immuneelectron microscopy of a 27-nm particle associated with acuteinfectious nonbacterial gastroenteritis. J. Virol. 10:1075-1081.

188. Kapikian, A. Z., R. G. Wyatt, H. B. Greenberg, A. R. Kalica,H. W. Kim, C. D. Brandt, W. J. Rodriguez, R. H. Parrott, andR. M. Chanock. 1980. Approaches to immunization of infantsand young children against gastroenteritis due to rotaviruses.Rev. Infect. Dis. 2:459-469.

189. Kaplan, J. E., G. W. Gary, R. C. Baron, N. Singh, L. B.Schonberger, R. Feldman, and H. B. Greenberg. 1982. Epi-demiology of Norwalk gastroenteritis and the role of Norwalkvirus in outbreaks of acute nonbacterial gastroenteritis. Ann.Intern. Med. 96:756-761.

190. Keswick, B. H., T. W. Hejkal, H. L. DuPont, and L. K.Pickering. 1983. Evaluation of a commercial enzyme immu-noassay kit for rotavirus detection. Diagn. Microbiol. Infect.Dis. 1:111-115.

191. Keswick, B. H., L. K. Pickering, H. L. DuPont, and W. E.Woodward. 1983. Prevalence of rotavirus in children in daycare centers. J. Pediatr. 103:85-86.

192. Kidd, A. H. 1984. Genome variants of adenovirus 41 (subgroupG) from children with diarrhoea in South Africa. J. Med. Virol.14:49-59.

193. Kidd, A. H., J. E. Banatvala, and J. C. de Jong. 1983.Antibodies to fastidious faecal adenoviruses (species 40 and41) in sera from chidren. J. Med. Virol. 11:333-341.

194. Kidd, A. H., F. E. Berkowtiz, P. J. Blaskovic, and B. D.Schoub. 1984. Genome variants of human adenovirus 40 (sub-group F). J. Med. Virol. 14:235-246.

195. Kidd, A. H., B. D. Cosgrove, R. A. Brown, and C. R. Madeley.1982. Faecal adenoviruses from Glasgow babies. Studies onculture and identity. J. Hyg. 88:463-474.

196. Kidd, A. H., E. H. Harley, and M. J. Erasmus. 1985. Specificdetection and typing of adenovirus types 40 and 41 in stoolspecimens by dot-blot hybridization. J. Clin. Microbiol. 22:934-939.

197. Kidd, A. H., and C. R. Madeley. 1981. In vitro growth of somefastidious adenoviruses from stool specimens. J. Clin. Pathol.34:213-216.

198. Kidd, A. H., A. Rosenblatt, T. G. Besselaar, M. J. Erasmus,C. T. Tiemessen, F. E. Berkowtiz, and B. D. Schoub. 1986.Characterization of rotaviruses and subgroup F adenovirusesfrom acute summer gastroenteritis in South Africa. J. Med.Virol. 18:159-168.

VOL. 2, 1989


r.asm.org/

Dow

nloaded from

http://cmr.asm.org/

84 CHRISTENSEN

199. Kjeldsberg, E. 1977. Small spherical viruses in faeces fromgastroenteritis patients. Acta Pathol. Microbiol. Scand. Sec. B85:351-354.

200. Kjeldsberg, E. 1985. Specific labelling of human rotavirusesand adenoviruses with gold-IgG complexes. J. Virol. Methods12:47-57.

201. Kjeldsberg, E., and J. C. Siebke. 1985. Use of immunosorbentelectron microscopy for detection of rota- and hepatitis A virusin sucrose solutions. J. Virol. Methods 12:161-167.

202. Knisley, C. V., A. J. Bednarz-Prashad, and L. K. Pickering.1986. Detection of rotavirus in stool specimens with monoclon-al and polyclonal antibody-based assay systems. J. Clin. Mi-crobiol. 23:897-900.

203. Konno, T., H. Suzuki, A. Imai, T. Kutsuzawa, N. Ishida, N.Katsushima, M. Sakamoto, S. Kitaoka, R. Tsuboi, and M.Adachi. 1978. A long-term survey of rotavirus infection inJapanese children with acute gastroenteritis. J. Infect. Dis.158:569-576.

204. Konno, T., H. Suzuki, M. Ishida, R. Chiba, K. Mochizuki, andA. Tsunoda. 1982. Astrovirus-associated epidemic gastroenter-itis in Japan. J. Med. Virol. 9:11-17.

205. Konno, T., H. Suzuki, N. Katsushima, A. Imai, F. Tazawa, T.Kutsuzawa, S. Kitaoka, M. Sakamoto, N. Yazaki, and N.Ishida. 1983. Influence of temperature and relative humidity onhuman rotavirus infection in Japan. J. Infect. Dis. 147:125-128.

206. Koopman, J. S., V. F. Turkisk, A. S. Monto, F. E. Thompson,and R. E. Isaacson. 1984. Milk fat and gastrointestinal illness.Am. J. Public Health 74:1371-1373.

207. Kouvelos, K., M. Petric, and P. J. Middleton. 1984. Compari-son of bovine, simian and human rotavirus structural glyco-proteins. J. Gen. Virol. 65:1211-1214.

208. Krause, P. J., J. S. Hyams, P. J. Middleton, V. C. Herson, andJ. Flores. 1983. Unreliability of Rotazyme ELISA test inneonates. J. Pediatr. 103:259-262.

209. Kurtz, J. B., and T. W. Lee. 1984. Human astrovirus se-rotypes. Lancet ii:1405.

210. Kurtz, J. B., T. W. Lee, and D. Pickering. 1977. Astrovirusassociated gastroenteritis in a children's ward. J. Clin. Pathol.30:948-952.

211. Kutsuzawa, T., T. Konno, H. Suzuki, A. Z. Kapikian, T. Ebina,and N. Ishida. 1982. Isolation of human rotavirus subgroups 1and 2 in cell culture. J. Clin. Microbiol. 16:727-730.

212. Laemmli, U. K. 1970. Cleavage of structural proteins duringassembly of the head of bacteriophage T4. Nature (London)227:680-685.

213. Lee, T. W. and J. B. Kurtz. 1981. Serial propagation ofastrovirus in tissue culture with the aid of trypsin. J. Gen.Virol. 57:421-424.

214. Lee, T. W. and J. B. Kurtz. 1982. Human astrovirus serotypes.J. Hyg. 89:539-540.

215. Leite, J. P. G., H. G. Pereira, R. S. Azeredo, and H. G.Schatzmayr. 1985. Adenoviruses in faeces of children withacute gastroenteritis in Rio De Janeiro, Brazil. J. Med. Virol.15:203-209.

216. Lewis, H. M., J. V. Parry, H. A. Davies, R. P. Parry, A. Mott.R. R. Dourmashkin, P. J. Sanderson, D. A. J. Tyrrell, and H. B.Valman. 1979. A year's experience of the rotavirus syndromeand its association with respiratory illness. Arch. Dis. Child.54:339-346.

217. Lin, M., M. Imai, A. R. Bellamy, N. Ikegami, Y. Furuichi, D.Summers, D. L. Nuss, and R. Deibel. 1985. Diagnosis ofrotavirus infection with cloned cDNA copies of viral genomesegments. J. Virol. 55:509-512.

218. Listernick, R., E. Zieserl, and A. T. Davis. 1986. Outpatientoral rehydration in the United States. Am. J. Dis. Child. 140:211-215.

219. Liu, M., P. A. Offit, and M. K. Estes. 1988. Identification of thesimian rotavirus SA11 genome segment 3 product. Virology163:26-32.

220. Liu, S., C. Birch, A. Coulepis, and I. Gust. 1984. Radioimmu-nofocus assay for detection and quantitation of human rota-virus. J. Clin. Microbiol. 20:347-350.

221. Lloyd-Evans, N., V. S. Springthorpe, and S. A. Sattar. 1986.

Chemical disinfection of human rotavirus-contaminated inani-mate surfaces. J. Hyg. 97:163-173.

222. Lopez, S., C. F. Arias, J. R. Bell, J. H. Strauss, and R. T.Espejo. 1985. Primary structure of the cleavage site associatedwith trypsin enhancement of rotavirus SA11 infectivity. Virol-ogy 144:11-19.

223. Losonsky, G. A., J. P. Johnson, J. A. Winkelstein, and R. H.Yolken. 1985. Oral administration of human serum immuno-globulin in immunodeficient patients with viral gastroenteritis.J. Clin. Invest. 76:2362-2367.

224. Losonsky, G. A., M. B. Rennels, A. Z. Kapikian, K. Midthun,P. J. Ferra, D. N. Fortier, K. M. Hoffman, A. Baig, and M. M.Levine. 1986. Safety, infectivity, transmissibility and immuno-genicity of rhesus rotavirus vaccine (MMU 18006) in infants.Pediatr. Infect. Dis. 5:25-29.

225. Losonsky, G. A., S. L. Vonderfecht, J. Eiden, S.-B. Wee, andR. H. Yolken. 1986. Homotypic and heterotypic antibodies forprevention of experimental rotavirus gastroenteritis. J. Clin.Microbiol. 24:1041-1044.

226. Maass, G., H. G. Bauneister, R. Heigemoller, P. Jansen, and H.Schumacher. 1978. Visualization by electron microscopy of23-nm virus particles (astrovirus) in faeces in newborns withacute nonbacterial gastroenteritis. Zentralbl. Bakteriol. Para-sitenkd. Infektionskr. Hyg. Abt. 1 Orig. Reihe A 242:423-430.

227. Madeley, C. R. 1979. Comparison of the features of astrovi-ruses and caliciviruses seen in samples of feces by electronmicroscopy. J. Infect. Dis. 139:519-523.

228. Madeley, C. R. 1986. The emerging role of adenoviruses asinducers of gastroenteritis. Pediatr. Infect. Dis. 5:S63-S73.

229. Madeley, C. R., and B. P. Cosgrove. 1975. Viruses in infantilegastroenteritis. Lancet ii:124.

230. Madeley, C. R., and B. P. Cosgrove. 1975. 28-nm particles infaeces in infantile gastroenteritis. Lancet ii:451-452.

231. Madeley, C. R., and B. P. Cosgrove. 1976. Caliciviruses in man.Lancet i:199-200.

232. Madeley, C. R., B. P. Cosgrove, E. J. Bell, and R. J. Fallon.1977. Stool viruses in babies in Glasgow. 1. Hospital admis-sions with diarrhea. J. Hyg. 78:261-273.

233. Madore, H. P., J. J. Treanor, and R. Dolin. 1986. Characteri-zation of the Snow Mountain agent of viral gastroenteritis. J.Virol. 58:487-492.

234. Madore, H. P., J. J. Treanor, K. A. Pray, and R. Dolin. 1986.Enzyme-linked immunosorbent assays for Snow Mountain andNorwalk agents of viral gastroenteritis. J. Clin. Microbiol. 24:456-459.

235. Maiya, P. P., S. M. Pereira, M. Mathan, P. Bhat, M. J. Albert,and S. J. Baker. 1977. Aetiology of gastroenteritis in infancyand early childhood in southern India. Arch. Dis. Child. 52:482-485.

236. Maki, M. 1981. A prospective clinical study of rotavirusdiarrhoea in young children. Acta Paediatr. Scand. 70:107-113.

237. Mankikar, S. D., P. J. Middleton, and M. Petric. 1979. Coun-terimmunoelectrophoresis assay for detection of adenovirusantigen. J. Clin. Microbiol. 10:253-255.

238. Marrie, T. J., S. H. S. Lee, R. S. Faulkner, J. Ethier, and C. H.Young. 1982. Rotavirus infection in a geriatric population.Arch. Intern. Med. 142:313-316.

239. Martin, M. L., G. W. Gary, Jr., and E. L. Palmer. 1979.Comparison of hemagglutination-inhibition, complement-fixa-tion, and enzyme-linked immunosorbent assay for quantitationof human rotavirus antibodies. Arch. Virol. 62:131-136.

240. Mason, B. B., D. Y. Graham, and M. K. Estes. 1980. In vitrotranscription and translation of simian rotavirus SA11 geneproducts. J. Virol. 3:1111-1121.

241. Mason, B. B., D. Y. Graham, and M. K. Estes. 1983. Biochem-ical mapping of the simian rotavirus SA11 genome. J. Virol. 46:413-423.

242. Matsumo, S., A. Hasegawa, A. Mukoyama, and S. Inouye.1985. A candidate for a new serotype of human rotavirus. J.Virol. 54:623-624.

243. Matsumo, S., S. Inouye, A. Hasegawa, and R. Kono. 1982.Assay of human rotavirus antibody by immune adherencehemagglutination with a cultivable human rotavirus as antigen.



r.asm.org/

Dow

nloaded from

http://cmr.asm.org/


J. Clin. Microbiol. 15:163-165.244. McAllister, R. M., M. 0. Nicolson, G. Reed, J. Kern, R. V.

Gilden, and R. J. Huebner. 1969. Transformation of rodentcells by adenovirus 19 and other group D adenovirus. J. Nati.Cancer Inst. 43:917-923.

245. McLean, B., and I. H. Holmes. 1980. Transfer of antirotaviralantibodies from mothers to their infants. J. Clin. Microbiol. 12:320-325.

246. McLean, B., S. Sonza, and I. H. Holmes. 1980. Measurement ofimmunoglobulin A, G, and M class rotavirus antibodies inserum and mucosal secretions. J. Clin. Microbiol. 12:314-319.

247. McLean, B. S., and I. H. Holmes. 1981. Effects of antibodies,trypsin, and trypsin inhibitors on susceptibility of neonates torotavirus infection. J. Clin. Microbiol. 13:22-29.

248. McSwiggan, D. A., D. Cubitt, and W. Moore. 1978. Calicivirusassociated with winter vomiting disease. Lancet i:1215

249. Middleton, P. J., M. D. Holdaway, M. Petric, M. T. Szymanski,and J. S. Tam. 1977. Solid-phase radioimmunoassay for thedetection of rotavirus. Infect. Immun. 14:439-444.

250. Middleton, P. J., M. Petric, C. M. Hewitt, M. T. Szymanski,and J. S. Tam. 1975. Counter-immunoelectro-osmophoresis forthe detection of infantile gastroenteritis virus in conventionaland germ-free piglets. Infect. Immun. 12:1276-1280.

251. Middleton, P. J., M. Petric, C. M. Hewitt, M. T. Szymanski,and J. S. Tam. 1976. Counter-immunoelectro-osmophoresis forthe detection of infantile gastroenteritis virus (Orbi-group)antigen and antibody. J. Clin. Pathol. 29:191-197.

252. Middleton, P. J., M. T. Szymanski, G. D. Abbott, R. Bortolussi,and J. R. Hamilton. 1974. Orbivirus acute gastroenteritis ofinfancy. Lancet i:1241-1244.

253. Middleton, P. J., M. T. Szymanski, and M. Petric. 1977.Viruses associated with acute gastroenteritis in young chil-dren. Am. J. Dis. Child. 131:733-737.

254. Midthun, K., H. B. Greenberg, T. Hoshino, A. Z. Kapikian,R. G. Wyatt, and R. M. Chanock. 1985. Reassortant rotavi-ruses as potential live rotavirus vaccine candidates. J. Virol.53:949-954.

255. Midthun, K., Y. Hoshino, A. Z. Kapikian, and R. M. Chanock.1986. Single gene substitution rotavirus reassortants containingthe major neutralization protein (VP7) of human rotavirusserotype 4. J. Clin. Microbiol. 24:822-826.

256. Miotti, P. G., J. Eiden, and R. H. Yolken. 1985. Comparativeefficiency of commercial immunoassays for the diagnosis ofrotavirus gastroenteritis during the course of infection. J. Clin.Microbiol. 22:693-698.

257. Moffet, H. L., H. K. Shulenberger, and E. R. Burkholder. 1968.Epidemiology and etiology of severe infantile diarrhea. J.Pediatr. 72:1-14.

258. Moosai, R. B., R. Alcock, T. M. Bell, F. R. Laidler, J. S. M.Peiris, A. P. Wyn-Jones, and C. R. Madeley. 1985. Detection ofrotavirus by a latex agglutination test Rotalex: comparisonwith electron microscopy, immunofluorescence, polyacryl-amide gel electrophoresis, and enzyme linked immunosorbentassay. J. Clin. Pathol. 38:694-700.

259. Moosai, R. B., M. J. Carter, and C. R. Madeley. 1984. Rapiddetection of enteric adenovirus and rotavirus: a simple methodusing polyacrylamide gel electrophoresis. J. Clin. Pathol. 37:1404-1408.

260. Mortensen, M. L., C. G. Ray, C. M. Payne, A. D. Friedman,L. L. Minnich, and C. Rousseau. 1985. Coronaviruslike parti-cles in human gastrointestinal disease. Am. J. Dis. Child. 139:928-934.

261. Murphy, A. M., M. B. Albrey, and E. B. Crewe. 1977.Rotavirus infections of neonates. Lancet ii:1149-1150.

262. Nagayoski, S., H. Yamaguchi, T. Ichikawa, M. Miyazu, T.Morishima, T. Ozaki, S. Isomura, S. Suzuki, and M. Hoshino.1980. Changes of the rotavirus concentration in faeces duringthe course of acute gastroenteritis as determined by the im-mune adherence hemagglutination test. Eur. J. Pediatr. 134:99-102.

263. 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.

Microbiol. 22:519-521.264. 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.

265. 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.

266. Nakata, S., M. K. Estes, and S. Chiba. 1988. Detection ofhuman calicivirus antigen and antibody by enzyme-linkedimmunosorbent assays. J. Clin. Microbiol. 26:2001-2005.

267. 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.

268. Nakata, S., B. L. Petrie, E. P. Calomeni, and M. K. Estes. 1987.Electron microscopy procedure influences detection of rotavi-ruses. J. Clin. Microbiol. 25:1902-1906.

269. Nicolaieff, A., G. Obert, and M. H. V. van Regenmortel. 1980.Detection of rotavirus by serological trapping on antibody-coated electron microscope grids. J. Clin. Microbiol. 12:101-104.

270. Nicolas, J. C., J. Cohen, B. Fortier, M. H. Lourenco, and F.Bricout. 1983. Isolation of an human pararotavirus. Virology124:181-184.

271. Niel, C., S. A. Gomes, J. P. G. Leite, and H. G. Pereira. 1986.Direct detection and differentiation of fastidious and nonfasti-dious adenovirus in stools by using a specific nonradioactiveprobe. J. Clin. Microbiol. 24:785-789.

272. Offit, P. A., and G. Blavat. 1986. Identification of the tworotavirus genes determining neutralization specificities. J. Vi-rol. 57:376-378.

273. Offit, P. A., G. Blavat, H. B. Greenberg, and H. F. Clark. 1986.Molecular basis of rotavirus virulence: role of gene segment 4.J. Virol. 57:46-49.

274. Offit, P. A., and H. F. Clark. 1985. Maternal antibody-medi-ated protection against gastroenteritis due to rotavirus innewborn mice is dependent on both serotype and titer ofantibody. J. Infect. Dis. 152:1152-1158.

275. Offit, P. A., H. F. Clark, W. G. Stroop, E. M. Twist, and S. A.Plotkin. 1983. The cultivation of human rotavirus, strain 'Wa,'to high titer in cell culture and characterization of the viralstructural polypeptides. J. Virol. Methods 7:29-40.

276. Offit, P. A., R. D. Shaw, and H. B. Greenberg. 1986. Passiveprotection against rotavirus-induced diarrhea by monoclonalantibodies to surface proteins vp3 and vp7. J. Virol. 58:700-703.

277. Orstavik, I., K. J. Figenschau, and J. C. Ulstrup. 1974. "Ro-tavirus" in stored specimens of faecal extracts. Lancet ii:1083.

278. Oshiro, L. S., C. E. Haley, R. R. Roberto, J. L. Riggs, M.Croughlan, H. Greenberg, and A. Kapikian. 1981. A 27-nmvirus isolated during an outbreak of acute infectious nonbac-terial gastroenteritis in a convalescent hospital: a possible newserotype. J. Infect. Dis. 143:791-796.

279. Pai, C. H., and D. Mayock. 1985. Rotazyme test in neonates. J.Pediatr. 106:343-344.

280. Pai, C. H., M. S. Shahrabadi, and B. Ince. 1985. Rapiddiagnosis of rotavirus gastroenteritis by a commercial latexagglutination test. J. Clin. Microbiol. 22:846-850.

281. Parrino, T. A., D. S. Schreiber, J. S. Trier, A. Z. Kapikian, andN. R. Blacklow. 1977. Clinical immunity in acute gastroenteritiscaused by Norwalk agent. N. Engl. J. Med. 297:86-89.

282. Paul, M. O., and E. A. Erinle. 1982. Influence of humidity onrotavirus prevalence among Nigerian infants and young chil-dren with gastroenteritis. J. Clin. Microbiol. 15:212-215.

283. Payne, C. M., and C. G. Ray. 1984. Coronavirus? Pediatrics74:560-562.

284. Payne, C. M., C. G. Ray, V. Borduin, L. L. Minnich, and M. D.Lebowitz. 1986. An eight-year study of the viral agents of acutegastroenteritis in humans: ultrastructural observations andseasonal distribution with a major emphasis on coronavirus-like particles. Diagn. Microbiol. Infect. Dis. 5:39-54.

285. Pedley, S., J. C. Bridger, J. F. Brown, and M. A. McCrae.1983. Molecular characterization of rotaviruses with distinct

VOL. 2, 1989


r.asm.org/

Dow

nloaded from

http://cmr.asm.org/

86 CHRISTENSEN

group antigens. J. Gen. Virol. 64:2093-2101.286. Pedley, S., J. C. Bridger, D. Chasey, and M. A. McCrae. 1986.

Definition of two new groups of atypical rotaviruses. J. Gen.Virol. 67:131-137.

287. Pereira, H. G., R. S. Azeredo, J. P. G. Leite, Z. P. Andrade,and L. De Castro. 1985. A combined enzyme immunoassay forrotavirus and adenovirus (EIARA). J. Virol. Methods 10:21-28.

288. Petric, M., P. J. Middleton, and S. D. Mankikar. 1979. Detec-tion of adenovirus antibody by counterimmunoelectropho-resis. J. Clin. Microbiol. 10:256-258.

289. Petrie, B. L., H. B. Greenberg, B. Y. Graham, and M. K. Estes.1984. Ultrastructural localization of rotavirus antigens usingcolloidal gold. Virus Res. 1:133-152.

290. Pina, M., and M. Green. 1965. Biochemical studies on adeno-virus multiplication. IX. Chemical and base composition anal-ysis of 28 human adenoviruses. Proc. Nati. Acad. Sci. USA 54:547-551.

291. Prince, S. D., C. Astry, S. Vonderfecht, G. Jakab, F.-M. Shen,and R. H. Yolken. 1986. Aerosol transmission of experimentalrotavirus infection. Pediatr. Infect. Dis. 5:218-222.

292. Puerto, F. I., L. Padilla-Noriega, A. Zamora-Chavez, A. Bri-ceno, M. Puerto, and C. F. Arias. 1987. Prevalent patterns ofserotype-specific seroconversion in Mexican children infectedwith rotavirus. J. Clin. Microbiol. 25:960-963.

293. Radetsky, M. 1986. Laboratory evaluation of acute diarrhea.Pediatr. Infect. Dis. 5:230-238.

294. Rand, K. H., H. J. Houck, and H. M. Swingle. 1985. Rotazymeassay in neonates without diarrhea. Am. J. Clin. Pathol. 84:748-751.

295. Ratnam, S., A. M. Tobin, J. B. Flemming, and P. J. Blaskovic.1984. False positive Rotazyme results. Lancet i:345-346.

296. Resta, S., J. P. Luby, C. R. Rosenfeld, and J. D. Siegel. 1985.Isolation and propagation of a human enteric coronavirus.Science 229:978-981.

297. Retter, M., P. J. Middleton, J. S. Tam, and M. Petric. 1979.Enteric adenoviruses: detection, replication, and significance.J. Clin. Microbiol. 10:574-578.

298. Rettig, P. J., and G. P. Altshuler. 1985. Fatal gastroenteritisassociated with coronaviruslike particles. Am. J. Dis. Child.139:245-248.

299. Reynolds, D., and J. H. Hughes. 1985. Comparison of theRotizyme assay with an avidin-biotin-amplified dot-immuno-binding assay for detecting rotaviruses. J. Infect. Dis. 152:647-648.

300. Richmond, S. J., S. M. Dunn, E. O. Caul, C. R. Ashley, andS. K. R. Clarke. 1979. An outbreak of gastroenteritis in youngchildren caused by adenoviruses. Lancet i:1178-1180.

301. Riepenhoff-Talty, M., S. Bogger-Goren, P. Li, P. J. Carmody,H. J. Barrett, and P. L. Ogra. 1981. Development of serum andintestinal antibody response to rotavirus after naturally ac-quired rotavirus infection in man. J. Med. Virol. 8:215-222.

302. 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.

303. Riepenhoff-Talty, M., H. Suzuki, and P. L. Ogra. 1983. Char-acteristics of the cell-mediated immune response to rotavirusin suckling mice. Dev. Biol. Stand. 53:263-268.

304. Rodger, S. M., R. F. Bishop, C. Birch, B. McLean, and I. H.

Holmes. 1981. Molecular epidemiology of human rotaviruses inMelbourne, Australia, from 1973 to 1979, as determined byelectrophoresis of genome ribonucleic acid. J. Clin. Microbiol.13:272-278.

305. Rodger, S. M., R. F. Bishop, and I. H. Holmes. 1982. Detectionof a rotavirus-like agent associated with diarrhea in infants. J.Clin. Microbiol. 16:724-726.

306. Rodriguez, W. J., H. W. Kim, C. D. Brandt, M. K. Gardner,and R. H. Parrott. 1983. Use of electrophoresis of RNA fromhuman rotavirus to establish the identity of strains involved inoutbreaks in a tertiary care nursery. J. Infect. Dis. 148:34-40.

307. Rodriguez, W. J., H. W. Kim, C. D. Brandt, R. H. Yolken, M.Richard, J. 0. Arrobio, R. H. Schwartz, A. Z. Kapikian, R. M.

Chanock, and R. H. Parrott. 1979. Common exposure outbreakof gastroenteritis due to type 2 rotavirus with high secondaryattack rate within families. J. Infect. Dis. 140:353-357.

308. Rosen, L. 1960. Hemagglutination inhibition technique fortyping adenovirus. Am. J. Hyg. 71:120-128.

309. Rotbart, H. A., R. H. Yolken, W. L. Nelson, D. Davis, M. H.Roe, and M. J. Levin. 1985. Confirmatory testing of Rotazymeresults in neonates. J. Pediatr. 107:289-292.

310. Rubenstein, A. S., and M. F. Miller. 1982. Comparison of anenzyme immunoassay with electron microscopic proceduresfor detecting rotavirus. J. Clin. Microbiol. 15:938-944.

311. Rudd, R. T., and D. Carrington. 1984. A prospective study ofchlamydial, mycoplasmal, and viral infections in a neonatalintensive care unit. Arch. Dis. Child. 59:120-125.

312. Sakuma, Y., S. Chiba, R. Kogasaka, H. Terashima, S. Naka-mura, K. Horino, and T. Nakao. 1981. Prevalence of antibodyto human calicivirus in general population of Northern Japan.J. Med. Virol. 7:221-225.

313. Sambourg, M., A. Goudeau, C. Courant, G. Pinon, and F.Denis. 1985. Direct appraisal of latex agglutination testing, aconvenient alternative to enzyme immunoassay for the detec-tion of rotavirus in childhood gastroenteritis, by comparison oftwo enzyme immunoassays and two latex tests. J. Clin. Micro-biol. 21:622-625.

314. Sanekata, T., and H. Okada. 1983. Human rotavirus detectionby agglutination of antibody-coated erythrocytes. J. Clin.Microbiol. 17:1141-1147.

315. Sanekata, T., Y. Toshida, and K. Oda. 1979. Detection ofrotavirus from faeces by reverse passive haemagglutinationmethod. J. Clin. Pathol. 32:963.

316. Sanekata, T., Y. Yoshida, K. Oda, and H. Okada. 1982.Detection of rotavirus antibody by inhibition of reverse passivehemagglutination. J. Clin. Microbiol. 15:148-155.

317. Santosham, M., R. H. Yolken, E. Quiroz, L. Dillman, G. Oro,W. C. Reeves, and R. B. Sack. 1983. Detection of rotavrius inrespiratory secretions of children with pneumonai. J. Pediatr.103:583-585.

318. Sarkkinen, H. K. 1981. Human rotavirus antigen detection byenzyme-immunoassay with antisera against Nebraska calf di-arrhoea virus. J. Clin. Pathol. 34:680-685.

319. Sarkkinen, H. K., H. Tuokko, and P. E. Halonen. 1980.Comparison of enzyme-immunoassay and radioimmunoassayfor detection of human rotaviruses and adenoviruses fromstool specimens. J. Virol. Methods 1:331-341.

320. Sato, K., Y. Inaba, T. Shinozaki, R. Fujii, and M. Matsumoto.1981. Isolation of human rotavirus cell cultures. Arch. Virol.69:155-160.

321. Schnagl, R. D., F. Morey, and I. H. Holmes. 1979. Rotavirusand coronavirus-like particles in aboriginal and non-aboriginalneonates in Kalgoorlie and Alice Springs. Med. J. Aust. 2:178-179.

322. Schoub, B. D. 1981. Enteric adenoviruses and rotaviruses ininfantile gastroenteritis in developing countries. Lancet ii:925.

323. Schoub, B. D., F. Cohen, D. Thompson, H. J. Koornhof, M. D.Miliotis, C. S. Still, F. E. Berkowitz, S. Miller, and E. Kushlick.1982. Variance in rotavirus infection rates in different urbanpopulation groups in South Africa. J. Med. Virol. 10:171-179.

324. Schoub, B. D., H. J. Koornhof, G. Lecatsas, 0. W. Prozesky, I.Freiman, E. Hartman, and H. Kassel. 1975. Viruses in acutesummer gastroenteritis in black infants. Lancet i:1093-1094.

325. Schreiber, D. S., N. R. Blacklow, and J. S. Trier. 1974. Thesmall intestinal lesion induced by Hawaii agent acute infectiousnonbacterial gastroenteritis. J. Infect. Dis. 129:705-708.

326. Shaw, R. D., K. J. Fong, G. A. Losonsky, M. M. Levine, Y.Maldonado, R. Yolken, J. Flores, A. Z. Kapikian, P. T. Vo, andH. B. Greenberg. 1987. Epitope-specific immune responses torotavirus vaccination. Gastroenterology 93:941-950.

327. Shaw, R. D., D. L. Stoner-Ma, M. K. Estes, and H. B.Greenberg. 1985. Specific enzyme-linked immunoassay forrotavirus serotypes 1 and 3. J. Clin. Microbiol. 22:286-291.

328. Shinozaki, T., K. Araki, H. Ushijima, and R. Fujii. 1987.Antibody response to enteric adenovirus types 40 and 41 insera from people in various age groups. J. Clin. Microbiol. 25:



r.asm.org/

Dow

nloaded from

http://cmr.asm.org/


1679-1682.329. Shinozaki, T., K. Araki, H. Ushijima, R. Fujii, and Y. Eshita.

1987. Use of Graham 293 cells in suspension for isolatingenteric adenoviruses from the stools of patients with acutegastroenteritis. J. Infect. Dis. 156:246.

330. Shinozaki, T., K. Araki, H. Ushijima, B. Kim, T. Tajima, andR. Fujii. 1986. Comparison of five methods for detectinghuman rotavirus in stool specimens. Eur. J. Pediatr. 144:513.

331. Shinozaki, T., K. Araki, H. Ushijima, B. Kim, T. Tajima, R.Fujii, and M. Minamitani. 1986. Coproantibody response torotavirus in an outbreak in a day-care nursery. Eur. J. Pediatr.144:515-516.

332. Singh, N., M. M. Sereno, J. Flores, and A. Z. Kapikian. 1983.Monoclonal antibodies to subgroup 1 rotavirus. Infect. Immun.42:835-837.

333. Singh-Naz, N., and R. K. Naz. 1986. Development and appli-cation of monoclonal antibodies for specific detection of hu-man enteric adenoviruses. J. Clin. Microbiol. 23:840-842.

334. Sitbon, M. 1985. Human-enteric-coronaviruslike particles(CVLP) with different epidemiological characteristics. J. Med.Virol. 16:67-76.

335. Smith, M. L., I. Lazdins, and I. H. Holmes. 1980. Codingassignments of double-stranded RNA segments of SA11 rota-virus established by in vitro translation. J. Virol. 33:976-982.

336. Soenarto, Y., T. Sebodo, R. Ridho, H. Alrasjid, J. E. Rohde,H. C. Bugg, G. L. Barnes, and R. F. Bishop. 1981. Acutediarrhea and rotavirus infection in newborn babies and chil-dren in Yogyakarta, Indonesia, from June 1978 to June 1979. J.Clin. Microbiol. 14:123-129.

337. Sonza, S., A. M. Breschkin, and I. H. Holmes. 1984. The majorsurface glycoproteins of simian rotavirus (SA11) containsdistinct epitopes. Virology 134:318-327.

338. Sonza, S., and I. H. Holmes. 1980. Coproantibody response torotavirus infection. Med. J. Aust. 2:496-499.

339. Spence, L., M. Fauvel, R. Petro, and S. Bloch. 1977. Compar-ison of counterimmunoelectrophoresis and electron micros-copy for laboratory diagnosis of human reovirus-like agentassociated with infantile gastroenteritis. J. Clin. Microbiol. 5:248-249.

340. Spencer, E., F. Avendano, and M. Araya. 1983. Characteristicsand analysis of electropherotypes of human rotavirus isolatedin Chile. J. Infect. Dis. 148:41-48.

341. Spratt, H. C., M. I. Marks, M. Gomersal, P. Gill, and C. H.Pai. 1978. Nosocomial infantile gastroenteritis associated withminirotavirus and calicivirus. J. Pediatr. 93:922-926.

342. Springthorpe, V. S., J. L. Grenier, N. Lloyd-Evans, and S. A.Sattar. 1986. Chemical disinfection of human rotaviruses:efficacy of commercially-available products in suspensiontests. J. Hyg. 97:139-161.

343. Stalhandske, P., R. Hyypia, A. Allard, P. Halonen, and U.Pettersson. 1985. Detection of adenoviruses in stool specimensby nucleic acid spot hybridization. J. Med. Virol. 16:213-218.

344. Stals, F., F. J. Walther, and C. A. Bruggeman. 1984. Faecaland pharyngeal shedding of rotavirus and rotavirus IgA inchildren with diarrhoea. J. Med. Virol. 14:333-339.

345. Suzuki, H., Y. Amano, H. Kinebuchi, E. G. Vera, A. Davila, J.Lopez, R. Gustabo, T. Konno, and N. Ishida. 1981. Rotavirusinfection in children with acute gastroenteritis in Ecuador. Am.J. Trop. Med. Hyg. 30:293-294.

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

347. Suzuki, H., T. Konno, Y. Numazaki, S. Kitaoka, T. Sato, A.Imai, F. Tazawa, T. Nakagomi, 0. Nakagomi, and N. Ishida.1984. Three different serotypes of human rotavirus determinedusing an interference test with coxsackie virus B1. J. Med.Virol. 13:41-44.

348. Suzuki, H., T. Sato, S. Kitaoka, F. Tazawa, T. Konno, Y.Amano, A. Alava-Alprecht, E. Gutierrez-Vera, J. Lopez-Vil-lalta, Y. Numazaki, and N. Ishida. 1986. Epidemiology ofrotavirus in Guayaquil, Ecuador. Am. J. Trop. Med. Hyg. 35:372-375.

349. Svensson, L., M. Grandien, and C.-A. Pettersson. 1983. Com-parison of solid-phase immune electron microscopy by use ofprotein A with direct electron microscopy and enzyme-linkedimmunosorbent assay for detection of rotavirus in stool. J.Clin. Microbiol. 18:1244-1249.

350. Svensson, L. 1984. Identification of an outer capsid glycopro-tein of human rotavirus by concanavalin A. J. Gen. Virol. 65:2183-2190.

351. Takiff, H. E., W. Reinhold, C. F. Garon, and S. E. Straus. 1984.Cloning and physical mapping of enteric adenovirus (candidatetype 40 and 41). J. Virol. 51:131-136.

352. Takiff, H. E., M. Seidlin, P. Krause, J. Rooney, C. Brandt, W.Rodriguez, R. Yolken, and S. E. Straus. 1985. Detection ofenteric adenoviruses by dot-blot hybridization using a molec-ularly cloned viral DNA probe. J. Med. Virol. 16:107-118.

353. Takiff, H. E., S. E. Straus, and C. F. Garon. 1981. Propagationand in vitro studies of previously non-cultivable enteral ade-noviruses in 293 cells. Lancet ii:832-834.

354. Tan, G. S., R. R. W. Townley, G. P. Davidson, R. F. Bishop,I. H. Holmes, and B. J. Ruck. 1974. Virus in faecal extractsfrom children with gastroenteritis. Lancet i:1109.

355. Taniguchi, K., S. Urasawa, and T. Urasawa. 1985. Preparationand characterization of neutralizing monoclonal antibodieswith different reactivity patterns to human rotaviruses. J. Gen.Virol. 66:1045-1053.

356. Thornhill, T. S., R. G. Wyatt, A. R. Kalica, R. Dolin, R. M.Chanock, and A. Z. Kapikian. 1977. Detection by immuneelectron microscopy of 26- to 27-nm viruslike particles associ-ated with two family outbreaks of gastroenteritis. J. Infect.Dis. 135:20-27.

357. Thouless, M. E., G. M. Beards, and T. H. Flewett. 1982.Serotyping and subgrouping of rotavirus strains by the ELISAtest. Arch. Virol. 73:219-230.

358. Thouless, M. E., A. S. Bryden, and T. H. Flewett. 1978.Serotypes of human rotavirus. Lancet i:39.

359. Thouless, M. E., A. S. Bryden, T. H. Flewett, G. N. Woode, J.C. Bridger, D. R. Snodgrass, and J. A. Herring. 1977. Serologicrelationships between rotaviruses from different species asstudied by complement-fixation and neutralization. Arch. Vi-rol. 53:287-294.

360. Tolia, V. K., and R. S. Dubois. 1985. Update of oral rehydra-tion: its place in treatment of acute gastroenteritis. Pediatr.Ann. 14:295-303.

361. Totterdell, B. M., J. E. Banatvala, and I. L. Chrystie. 1983.Studies on human lacteal rotavirus by immune electron micros-copy. J. Med. Virol. 11:167-175.

362. Totterdell, B. M., J. E. Banatvala, I. L. Chrystie, G. Ball, andW. D. Cubitt. 1988. Systemic lymphoproliferative responses torotavirus. J. Med. Virol. 25:37-44.

363. Totterdell, B. M., I. L. Chrystie, and J. E. Banatvala. 1980.Cord blood and breast-milk antibodies in neonatal rotavirusinfection. Br. Med. J. 280:828-830.

364. Totterdell, B. M., K. G. Nicholson, J. MacLeod, I. L. Chrystie,and J. E. Banatvala. 1982. Neonatal rotavirus infection: role oflacteal neutralising activity, alpha1 anti-trypsin and non-immu-noglobulin antiviral activity in protection. J. Med. Virol. 10:37-44.

365. Tronen, H. 1984. False positive Rotazyme results. Lancet i:345.

366. Tufvesson, B., and T. Johnsson. 1976. Immunoelectroosmo-phoresis for detection of reo-like virus: methodology andcomparison with electron microscopy. Acta Pathol. Microbiol.Scand. Sect. B 84:225-228.

367. Uhnoo, I., E. Olding-Stenkvist, and A. Kreuger. 1986. Clinicalfeatures of acute gastroenteritis associated with rotavirus,enteric adenoviruses, and bacteria. Arch. Dis. Child. 61:732-738.

368. Uhnoo, I., and L. Svensson. 1986. Clinical and epidemiologicalfeatures of acute infantile gastroenteritis associated with hu-man rotavirus subgroups 1 and 2. J. Clin. Microbiol. 23:551-555.

369. Uhnoo, I., G. Wadell, L. Svensson, and M. Johansson. 1983.Two new serotypes of enteric adenovirus causing infantile

VOL. 2, 1989


r.asm.org/

Dow

nloaded from

http://cmr.asm.org/

88 CHRISTENSEN

diarrhoea. Dev. Biol. Stand. 53:311-318.370. Uhnoo, I., G. Wadell, L. Svensson, and M. Johansson. 1984.

Importance of enteric adenoviruses 40 and 41 in acute gas-troenteritis in infants and young children. J. Clin. Microbiol.20:365-372.

371. Urasawa, S., T. Urasawa, and K. Taniguchi. 1982. Threehuman rotavirus serotypes demonstrated by plaque neutraliza-tion of isolated strains. Infect. Immun. 38:781-784.

372. Urasawa, T., S. Urasawa, and K. Taniguchi. 1981. Sequentialpassage of human rotavirus in MA-104 cells. Microbiol. Immu-nol. 25:1025-1035.

373. Ushijima, H., K. Bosu, T. Abe, and T. Shinozaki. 1986.Suspected rotavirus encephalitis. Arch. Dis. Child. 61:692-694.

374. van Loon, A. E., R. Maas, R. T. M. J. Vaessen, A. M. C. B.Reemst, J. S. Sussenbach, and T. H. Rozijn. 1985. Cell trans-formation by the left terminal regions of the adenovirus 40 and41 genomes. Virology 147:227-230.

375. van Loon, A. E., T. H. RoziJn, J. C. de Jong, and J. S.Sussenbach. 1985. Physicochemical properties of the DNAs ofthe fastidious adenovirus species 40 and 41. Virology 140:197-200.

376. Vesikari, T. 1985. Progress in rotavirus vaccination. Pediatr.Infect. Dis. 4:612-614.

377. Vesikari, T., E. Isolauri, A. Delem, E. D'Hondt, F. E. Andre,G. M. Beards, and T. H. Flewett. 1985. Clinical efficacy of theRIT 4237 live attenuated bovine rotavirus vaccine in infantsvaccinated before a rotavirus epidemic. J. Pediatr. 107:189-194.

378. Vesikari, T., E. Isolauri, A. Delem, E. D'Hondt, F. E. Andre,and G. Zissis. 1983. Immunogenicity and safety of live oralattenuated bovine rotavirus vaccine strain RIT 4237 in adultsand young children. Lancet ii:807-811.

379. Vesikari, T., E. Isolauri, E. D'Hondt, A. Delem, F. E. Andre,and G. Zissis. 1984. Protection of infants against rotavirusdiarrhoea by RIT 4237 attenuated bovine rotavirus strainvaccine. Lancet i:977-980.

380. Vesikari, T., A. Z. Kapikian, A. Delem, and G. Zissis. 1986. Acomparative trial of rhesus monkey (RRV-1) and bovine (RIT4237) oral rotavirus vaccines in young children. J. Infect. Dis.153:832-839.

381. Vesikari, T., M. Maki, H. J. Sarkkinen, P. P. Arstila, and P. E.Halonen. 1981. Rotavirus, adenovirus, and non-viral entero-pathogens in diarrhea. Arch. Dis. Child. 56:264-270.

382. Vesikari, T., T. Ruuska, H. Bogaerts, A. Delem, and F. Andre.1985. Dose-response study of RIT 4237 oral rotavirus vaccinein breast-fed and formula-fed infants. Pediatr. Infect. Dis. 4:622-625.

383. Voller, A., A. Bartlett, D. E. Bidwell, M. F. Clark, and A. N.Adams. 1976. The detection of viruses by enzyme-linkedimmunosorbent assay (ELISA). J. Gen. Virol. 33:165-167.

384. Vollett, J. J., III, H. L. DuPont, and L. K. Pickering. 1981.Nonenteric sources of rotavirus in acute diarrhea. J. Infect.Dis. 144:495.

385. von Bonsdorff, C.-H., T. Hovi, P. Makela, and A. Morttinen.1978. Rotavirus infections in adults in association with acutegastroenteritis. J. Med. Virol. 2:21-28.

386. Wadell, G. 1984. Molecular epidemiology of human adenovi-ruses. Curr. Top. Microbiol. Immunol. 110:191-220.

387. Wadell, G., M.-L. Hammarskjold, G. Winberg, T. M. Var-sanyi, and G. Sundell. 1980. Genetic variability of adenovi-ruses. Ann. N.Y. Acad. Sci. 354:16-42.

388. Wall, R. A., B. J. Mellars, P. Luton, and S. Boulding. 1982.Comparison of ELISA, SPACE, and electron microscopy forthe routine diagnosis of rotavirus infection. J. Clin. Pathol. 35:104-106.

389. Walther, F. J., C. Bruggeman, M. S. M. Daniels-Bosman, S.Pourier, G. Grauls, F. Stals, and A. V. D. Boggard. 1983.Symptomatic and asymptomatic rotavirus infections in hospi-talized children. Acta Paediatr. Scand. 72:659-663.

390. Ward, R. L., D. I. Bernstein, E. C. Young, J. R. Sherwood,D. R. Knowlton, and G. M. Schiff. 1986. Human rotavirusstudies in volunteers: determination of infectious dose and

serological response to infection. J. Infect. Dis. 154:871-884.391. Ward, R. L., D. R. Knowlton, and M. J. Pierce. 1984. Effi-

ciency of human rotavirus propagation in cell culture. J. Clin.Microbiol. 19:748-753.

392. Weinberg, R. J., G. Tipton, W. J. Klish, and M. R. Brown.1984. Effect of breast-feeding on morbidity in rotavirus gas-troenteritis. Pediatrics 74:250-253.

393. Weiss, C., and H. F. Clark. 1985. Rapid inactivation ofrotaviruses by exposure to acid buffer or acidic gastric juice. J.Gen. Virol. 66:2725-2730.

394. White, L., I. Perez, M. Perez, G. Urbina, H. Greenberg, A.Kapikian, and J. Flores. 1984. Relative frequency of rotavirussubgroups 1 and 2 in Venezuelan children with gastroenteritisas assayed with monoclonal antibodies. J. Clin. Microbiol. 19:516-520.

395. Whitelaw, A., H. Davies, and J. Parry. 1977. Electron micros-copy of fatal adenovirus gastroenteritis. Lancet i:361.

396. Williams, E. K., J. A. Lohr, and R. L. Guerrant. 1986. Acuteinfectious diarrhea. II. Diagnosis, treatment, and prevention.Pediatr. Infect. Dis. 5:458-465.

397. Wong, C. J., Z. Price, and D. A. Bruckner. 1984. Asepticmeningitis in an infant with rotavirus gastroenteritis. Pediatr.Infect. Dis. 3:244-246.

398. Wood, D. J., and A. S. Bailey. 1987. Detection of adenovirustypes 40 and 41 in stool specimens by immune electronmicroscopy. J. Med. Virol. 21:191-199.

399. Woode, G. N., J. C. Bridger, J. M. Jones, T. H. Flewett, A. S.Bryden, H. A. Davies, and G. B. B. White. 1976. Morphologicand antigenic relationships between viruses (rotaviruses) fromacute gastroenteritis of children, calves, piglets, mice, andfoals. Infect. Immun. 14:804-810.

400. Woode, G. N., N. E. K. Gourley, J. F. Pohlenz, E. M. Leibler,S. L. Marhews, and M. P. Hutchinson. 1985. Serotypes ofbovine astrovirus. J. Clin. Microbiol. 22:668-670.

401. Wyatt, R. G., R. Dolin, N. R. Blacklow, H. L. DuPont, R. F.Buscho, T. S. Thornhill, A. Z. Kapikian, and R. M. Chanock.1974. Comparison of three agents of acute infectious nonbac-terial gastroenteritis by cross-challenge in volunteers. J. Infect.Dis. 129:709-714.

402. Wyatt, R. G., H. B. Greenberg, W. D. James, A. L. Pittman,A. R. Kalica, J. Flores, R. M. Chanock, and A. Z. Kapikian.1982. Definition of human rotavirus serotypes by plaque reduc-tion assay. Infect. Immun. 37:110-115.

403. Wyatt, R. G., W. D. James, E. H. Bohl, K. W. Theil, L. J. Saif,A. R. Kalica, H. B. Greenberg, A. Z. Kapikian, and R. M.Chanock. 1980. Human rotavirus type 2: cultivation in vitro.Science 207:189-191.

404. Wyatt, R. G., H. D. James, Jr., A. L. Pittman, Y. Hoshino,H. B. Greenberg, A. R. Kalica, J. Flores, and A. Z. Kapikian.1983. Direct isolation in cell culture of human rotaviruses andtheir characterization into four serotypes. J. Clin. Microbiol.18:310-317.

405. Wyatt, R. G., A. R. Kalica, C. A. Mebus, H. W. Kim, W. T.London, R. M. Chanock, and A. Z. Kapikian. 1978. Reovirus-like agents (rotaviruses) associated with diarrheal illness inanimals and man. Perspect. Virol. 10:121-145.

406. Yamaguchi, H., S. Inouye, M. Yamauchi, T. Morishima, S.Matsuno, S. Isomura, and S. Suzuki. 1985. Anamnestic re-sponse in fecal IgA antibody production after rotaviral infec-tion of infants. J. Infect. Dis. 152:398-400.

407. Yolken, R. H. 1983. Avidin-biotin radioimmunoassay for hu-man rotavirus. J. Infect. Dis. 148:942.

408. Yolken, R. H., H. W. Kim, T. Clem, R. G. Wyatt, A. R. Kalica,R. M. Chanock, and A. Z. Kapikian. 1977. Enzyme-linkedimmunosorbent assay (ELISA) for detection of human reo-virus-like agent of infantile gastroenteritis. Lancet ii:263-266.

409. Yolken, R. H., F. Lawrence, F. Leister, H. E. Takiff, and S. E.Strauss. 1982. Gastroenteritis associated with enteric typeadenovirus in hospitalized infants. J. Pediatr. 101:21-26.

410. Yolken, R. H., and F. J. Leister. 1981. Evaluation of enzymeimmunoassays for the detection of human rotavirus. J. Infect.Dis. 144:379.

411. Yolken, R. H., G. A. Losonsky, S. Vonderfecht, F. Leister, and



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VIRAL GASTROENTERITIS

S.-B. Wee. 1985. Antibody to human rotavirus in cow's milk.N. Engl. J. Med. 312:605-610.

412. Yolken, R. H., L. Mata, B. Garcia, J. J. Urrutia, R. G. Wyatt,R. M. Chanock, and A. Z. Kapikian. 1978. Secretory antibodydirected against rotavirus in human milk-measurement bymeans of enzyme-linked immunosorbent assay (ELISA). J.Pediatr. 93:916-921.

413. Yolken, R., and M. Murphy. 1982. Sudden infant death syn-

drome associated with rotavirus infection. J. Med. Virol. 10:291-296.

414. Yolken, R. H., and P. J. Stopa. 1979. Analysis of nonspecificreactions in enzyme-linked immunosorbent assay testing forhuman rotavirus. J. Clin. Microbiol. 10:703-707.

415. Yolken, R. H., R. G. Wyatt, A. R. Kalica, H. W. Kim, C. D.Brandt, R. H. Parrott, A. Z. Kapikian, and R. M. Chanock.1977. Use of a free viral immunofluorescence assay to detecthuman reovirus-like agent in human stools. Infect. Immun. 16:467-470.

416. Yolken, R. H., R. G. Wyatt, and A. Z. Kapikian. 1977. ELISAfor rotavirus. Lancet ii:819.

417. Yolken, R. H., R. G. Wyatt, H. W. Kim, A. Z. Kapikian, andR. M. Chanock. 1978. Immunological response to infection

with human reovirus-like agent: measurement of anti-humanreovirus-like agent immunoglobulin G and M levels by themethod of enzyme-linked immunosorbent assay. Infect. Im-mun. 19:540-546.

418. Yolken, R. H., R. G. Wyatt, G. Zissis, C. D. Brandt, W. J.Rodriquez, H. W. Kim, R. H. Parrott, J. J. Urrutia, L. Mata,H. B. Greenberg, A. Z. Kapikian, and R. M. Chanock. 1978.Epidemiology of human rotavirus types 1 and 2 as studied byenzyme-linked immunosorbent assay. N. Engl. J. Med. 299:1156-1161.

419. Zentner, B.-S., M. Margalith, A. Galil, B. Halevy, and I. Sarov.1985. Detection of rotavirus-specific IgG antibodies by immu-noperoxidase assay and enzyme-linked immunosorbent assay.J. Virol. Methods 11:199-206.

420. Zissis, G., and J. P. Lambert. 1978. Different serotypes ofhuman rotavirus. Lancet i:38-39.

421. Zissis, G., and J. P. Lambert. 1980. Enzyme-linked immuno-sorbent assays adapted for serotyping of human rotavirusstrains. J. Clin. Microbiol. 11:1-5.

422. Zissis, G., J. P. Lambert, and D. Deicegel. 1978. Routinediagnosis of human rotavirus in stools. J. Clin. Pathol. 31:175-178.

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