Human Rotavirus Studies in Volunteers: Determination of Infectious Dose and Serological Response to Infection

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1 THE JOURNAL OF INFECTIOUS DISEASES VOL. 154, NO.5. NOVEMBER by The University of Chicago. All rights reserved /86/ $01.00 Human Rotavirus Studies in Volunteers: Determination of Infectious Dose and Serological Response to Infection Richard L. Ward, David I. Bernstein, Elizabeth C. Young, James R. Sherwood, Douglas R. Knowlton, and Gilbert M. Schiff From the James N. Gamble Institute of Medical Reasearch, Cincinnati, Ohio An unpassaged, safety-tested strain (CJN) of human rotavirus from a stool specimen of a hospitalized child was administered orally to 62 adult volunteers for determination of the dose required to produce infection with or without illness. Subjects ingested doses ranging from 9 X 10-3 to 9 X 10 4 focus-forming units in buffered salt solution after consumption of 50 ml of 4070 NaHC0 3 The amount of virus in the inoculum required to cause infection (shedding of virus, seroconversion, or both) in study subjects was comparable to the minimum detectable in cultures of primary monkey kidney cells. Seventeen of 30 infected subjects became ill with doses equivalent to that required for infection. Although the preinoculation titers of serum neutralizing antibody to the challenge virus in study subjects ranged from <1:2 to 1:1,600, the concentration of serum antibody could not be correlated with protection from infection or illness in subjects given an infectious dose of virus. Rotaviruses are recognized as the major cause of acute diarrheal disease in infants and young children [1]. Individuals infected with these viruses often excrete > particles/g of fecal matter [2, 3], and rotaviruses can survive for days under environmental conditions [4]. It should therefore be possible to transmit rotavirus disease from person to person through a variety ofenvironmental pathways, including contaminated water, provided the inoculum of ingested virus is sufficiently large. Waterborne outbreaks of rotavirus disease have been reported, of which the most notableoccurredin Chinain 1982 [5]. Previous studies with other enteric viruses have suggested a human infectious dose of between 1 and 919 pfu or TCID so [6, 7]. These studies were performed with attenuated polioviruses or echovirus type 12, neither of which caused illness under Received for publication 12September 1986,and in revisedform 25 April The data in this study have not been subjected to the required peer and administrative review of the U.S. Environmental Protection Agency and therefore do not necessarily reflect the views of the Agency, and no offical endorsement should be inferred. This study and the informed consent form used were approved by the Christ Hospital Institutional ReviewBoard and the University of Cincinnati College of Medicine Committee on Human Research. These studies were funded wholly or in part by assistance agreement R from the U.S. Environmental Protection Agency. Please address requests for reprints to Dr. Richard L. Ward, James N. Gamble Institute of Medical Research, 2141 Auburn Avenue, Cincinnati, Ohio the experimental conditions. Thus the relation between the dose of virus consumed and clinical illness was not determinable, and the dose causing illness could be considerably greater than that required to cause infection. Rotavirus infection, however, often results in illness. Determination of the dose required to cause both infection and illness should clarify the public health significance of consumption of small amounts ofthis virus, a situation that may occur with «ontaminated potable water. Although most severe rotavirus illnesses involve infants and young children, rotaviruses frequently cause illness in adults [1]. Thus adults should be suitable subjects for an infectious dose determination provided they are susceptible to the challenge strain of virus. Rotavirus disease in adults is usually the result of reinfection rather than primary infection because almost all adults have serological evidence of a previous infection. Reinfection may be possible because of loss of immunity or exposure to a different serotype of rotavirus. Ifeither of these explanations is valid, persons most susceptible to infection could be expected to have the least amount of neutralizing antibody to the challenge virus. A correlation between titer of serum neutralizing antibody and susceptibility to infection has been suggested from a study by Kapikian et al. [8] involving 18 adult subjects. It should be noted, however, that even the four subjects who became ill in that study had a geometric mean titer (GMT) of neutralizing antibody of 1:74 to the serotype of the challenge vi- 871

2 872 Ward et al. rus. Thus the presence of serum neutralizing antibody did not fully protect these volunteers against infection or illness. In the present study different doses of a safetytested preparation of human rotavirus from the stool of an infected child were given to adult volunteers to determine the number oftissue culture infectious particles required to cause both infection and illness. Because the adults selected had a wide range of titers of serum neutralizing antibody to the challenge virus, the effect of this antibody on the probability of infection and illness in study subjects was also determined. Materials and Methods Virus and cells. The rotavirus preparation used in this study was an unpassaged strain (CJN) obtained from the stool of an eight-month-old child hospitalized with acute gastroenteritis at Children's Hospital Medical Center in Cincinnati in May A 5070 suspension of stool in Earle's balanced salt solution was blended for 2 min and centrifuged at 8,000 g for 30 min. The supernatant was filtered (pore size 0.2 urn), and 2-ml aliquots were stored at - 70 C. The preparation was safety tested according to acceptable standards [9], which included a radioimmunofocus assay to detect hepatitis A virus [10] performed by Dr. Mark Sobsey (University of North Carolina, Chapel Hill). This preparation contained an average of 1.4 X 10 9 rotavirus particles/ml, >900;0 of which had the double protein shells required for infectivity [11], as quantified by a pseudoreplication technique [12] performed by Dr. Betty Petrie (Baylor College of Medicine, Houston). The preparation also contained 9 ± 3 x 10 4 focus-forming units (ffu)/ml, as measured in primary African green monkey kidney cells [3]. Thus its particle/ffu ratio was 1.56 x 10 4 The CJN virus and other strains of human rotavirus were adapted to grow in cell culture by two passages in primary African green monkey kidney cells as described previously [3]. These viruses and representatives of the four established human rotavirus serotypes were thengrown in MA-I04cells, also by methods already described [3]. Serotypes 1-4 were represented by strains Wa, DS-l, P, and ST-3, respectively [13], all of which were provided by Dr. Richard Wyatt (National Institutes of Health, Bethesda, Md). Electrophoretic analysis of rotavirus RNA. The electrophoretic properties of RNA genome seg- ments from different strains ofhuman rotavirus were determined by PAGE. RNA of the CJN virus was extracted with phenol directly from the stool sample as well as from the filtered stool preparation according to methods described by Pons [14]. RNA was also extracted from tissue culture-adapted preparations of the CJN virus and other human strains grown in MA-I04 cells. The equivalent of 0.25 ml of each preparation of virus was analyzed. Electrophoresis was performed in polyacrylamide slab gels (thickness 0.5 mm) at a constant current of 10 rna for 18 hr at room temperature (rv23 C) [3]. The positions of the RNA bands were determined by silver staining according to methods described by Dolan et al. [15]. Serotype determination ofcjn virus. Cell culture-adapted CJN virus and the Wa, DS-I, P, and ST-3strains of human rotavirus were grown in MA 104 cells, purified by CsCl gradient centrifugation, and used to hyperimmunize guinea pigs according to methods described previously [16]. After immunization ofthe animals, sera collected by heart puncture were heat inactivated at 56 C for 30 min, and neutralization titers were determined with a plaque assay in MA-I04 cells [16]. The neutralization titer is defined as the reciprocal of the serum dilution required to reduce the number of viral pfu by as determined by graphic interpolation of plaque assays. Study subjects. Participants in the study were healthy, year-old men. An initial blood specimen was collected from each subject for determination of plaque neutraliziation titer against a reassortant of the tissue culture-adapted CJN strain of human rotavirus (see Results for a detailed explanation). Subjects with initial titers of ~30were recalled, and a general physical examination, urinalysis, complete blood cell counts, and a battery of blood chemistry assays were done to ensure good health status. If a subject was in good health, the subject was enrolled in the study. Study plan. The study was divided into three segments. The first segment included six subjects who were given either of the two highest doses of virus used in the study: 9 x 10 4 ffu (three subjects) or 9 x 10 3 ffu (three subjects). The second segment included 26 subjects randomly assigned to receive one of four doses of virus ranging between 9 and 9 x 10 3 ffu and five subjects who were given placebo. The final segment included 25 subjects randomly given four doses of virus of between 9 x 10-3 and 9 ffu. Subjects were instructed to consume nothing

3 Human Rotavirus Studies in Volunteers 873 by mouth except water for 4 hr before and after consumptionofthe virus. Virus was administered in 50 ml of cold Earle's balanced salt solution after consumption of 50 ml of 4070 NaHC0 3 This procedure was used to protect the virus, which becomes partially inactivated in distilled water and below ph 5 (authors' unpublished data). After administration of virus (day 0), subjects were asked to consume no milk for 2 days to avoid potential effects of milk immunoglobulins on viral infection [17]. Subjects were admitted to the isolation facility two days after consumption of virus and remained for six days. The subjects generally slept two to a room but intermingled freely. All meals were eaten as a group. No visitors were permitted. No medication was permitted during the isolation period unless it was approved by study personnel. During the isolation period participants were examined twice a day for signs and symptoms of illness, which were scored according to the following scale: 0 = absent; 1 = mild; 2 = moderate; and 3 = severe. Vital signs (temperature and pulse) were recorded at least twice a day. A stool specimen was collected on the day that virus was given (before administration). All stools were collected during isolation, and one each was collected on all return visits (days 10-11, 13-14, and 26-28). Stool specimens were stored frozen at - 20 C. Blood specimens were collected on days 0 and 8 and on all return visits. Samples collected on day 8 and return visits were analyzed by a number of standard chemistry assays (e.g., serum glutamic oxaloacetic and pyruvic transaminases and blood urea nitrogen). Those specimens collected on days 0 and were tested for serum neutralizing antibody. A final physical examination was done on the last visit. Determination ofshedding of virus. The presence of virus in stool specimens was detected by an ELISA. The procedure was a modification ofthe assay described in the manual that accompanies the kit sold by Dakopatts A/S (Copenhagen). This ELISA incorporates the use of biotin-avidin complexes for increasing the limit of sensitivity, which was "-'10 6 rotavirus particles/ml of processed stool material. The procedure consisted of the following steps. A suspension of thawed stool material in Earle's balanced salt solution was blended for 2 min and centrifuged at 1,500 g for 20 min. The supernatant was frozen in 2-ml aliquots at - 20 C. Immediately before analysis an aliquotwas thawed, and EDTA was added to a final concentration of 2 mm to remove the outer protein shell and to better expose the rotavirus group antigen [18]. These samples (0.1 ml) were added to wells of microtiter plates coated with either rabbit antibody to rotavirus or preimmune rabbit serum (Dakopatts). After incubation for 1 hr, plates were washed with buffer (0.5 MNaCl, 1.5 rom KH2P04, 6.5 rom Na2HP04, and 0.05 Tween'" 20 [Sigma Chemical, St. Louis], and then normal goat serum (dilution, 1:50, Vector Laboratories, Burlingame, Calif) in PBS containing 5070 nonfat dry milk (PBS-M) was added (0.1 ml per well) and incubated for 30 min. Each well received an additionalo.1 ml of serum from a guinea pig hyperimmunized with the Wa strain of rotavirus (1:200 dilution in PBS-M with a 1:50 dilution ofnormal rabbit serum [Dakopatts]). After 30 min the plates were washed, and 0.1 ml of biotinylated goat antiserum to guinea pig IgG 1:100 dilution [Vector] in PBS-M containing a 1:50 dilution of normal rabbit serum) was added and incubated for 30 min before washing. Avidin preincubated for 30 min with peroxidaseconjugated biotin (1:50 dilution of each in wash buffer; Vector) was added (0.1 ml per well), incubated 30 min, and washed from the plates. After incubation for 30 min with 0.1 ml of substrate (a-phenylenediamine), the reaction was stopped with addition of 0.15 ml of 1 M H 2S04, and the colorimetric reaction was measured by A 4 90 with an automated reader (Litton Bionetics, Charleston, SC). Samples were considered positive if their A value was at least twice that of the controls and > Each sample was tested in duplicate. Determination ofseroconversion. The volunteers' titers of serum neutralizing antibody were determined as follows. Sera collected on days 0 and were heat inactivated, serially diluted twofold with Earle's balanced salt solution containing tryptose phosphate broth, and mixed with an equal volume of "-'1,000 pfu of tissue culture-adapted CJN virus/ml in the same buffer. After incubation ofthe samples at 37 C for 30 min, confluent monolayers of MA-I04 cells in 60-mm tissue culture plates were washed with Earle's balanced salt solution to remove serum. Survival of virus was then measured by plaque assay with use of 0.2 ml of sample per plate [16], and neutralization titers were calculated. A greater than twofold rise in titer between acute- and convalescent-phase sera was considered to be significant. These same methods were used to measure, in selected subjects, rises in titers of neutralizing antibody to representatives of the four established serotypes of human rotavirus.

4 874 Ward et al. Statistical analysis ofdata. Two types ofstatistical calculations were done during this study. The Kruskel-Wallis test [19] was used to determine the relation between the amount of shedding of virus and the extent of rise in antibody titer in infected subjects. The relation between the dose of the challenge CJN virus, as determined by a fluorescent focus assay in primary African green monkey kidney cells, and infectivity in humans was estimated by probit analysis [20]. Probit transformation was used because it is based on normal tolerance distribution; the assumption of normality seems most appropriate in the analysis of this type of information because differences in individual susceptibility are probably the primary reasons for differences in response among study subjects given the same dose. All statistical calculations were performed by Dr. Judy Stober (U.S. Environmental Protection Agency, Cincinnati). Results Properties ofchallenge virus. The CJN strain of human rotavirus used in this study was selected from among 16 candidates obtained from hospitalized children. This virus could be distinguished by electrophoretic analysis of RNA genome segments from the other rotavirus isolates. The electrophoretic patternof RNA from thecjn virus also was easily distinguishable from RNA patterns of rotaviruses representative of the four established human serotypes: Wa (serotype 1), DS-l (serotype 2), P (serotype 3), and ST-3 (serotype 4; figure 1). The original selection ofthe CJN isolate was based on the finding that the laboratory-grown preparation of this virus was among the least neutralized by 10 randomly selected adult sera. After the study was underway, we found that the tissue cultureadapted CJN isolate from the child's stool was actually a reassortant containing RNA segments from both the CJN virus and a second rotavirus presumed to be present in stool material in concentrations too low to be detected by electrophoretic analysis of viral RNA. The immediate concern was that more than one human rotavirus was also present in the filtered preparation of challenge virus that had been given to volunteers. This concern was alleviated when we found that human rotaviruses cultured from this challenge preparation (16 replicates) all contained RNA electrophoretic profiles identical to that obtained with virus present in the original stool, and Wa DS 1 P ST-3 CJN Serotype -- Figure 1. Schematic diagram of the electrophoretic profile of RNA segments from human rotaviruses representative of serotypes 1-4 and from CJN virus extracted directly from a stool sample. none had evidence ofadditional segments (figure 1). Because the filtration step used to make the challenge preparation removed "-'99070 of infectious viruses, filtration also apparently removed the small amount of the second rotavirus present in the original stool. Later we showed that volunteers given this challenge preparation shed only viruses whose RNA was also electrophoretically identicalto that detected in the originalstool from thechild. Furthermore, virus cultured from stool samples ofthese subjects had RNA electrophoretic profiles identical to that of the original CJN virus. Thus study subjects were apparently given only a single strain of virus. The serotypic relatedness of the CJN virus to representatives of the four established serotypes of human rotavirus was determined by two-way crossneutralization. Guinea pigs were hyperimmunized with each of the five viruses, and homologous and heterologous titers were measured. The CJN virus used in this and subsequent studies was a triply plaque-purified isolate obtained after cell culture adaptationofthe virus present in the challenge preparation. Its RNA electrophoretic profile was identical to that of virus detectable in the original stool. The CJN virus shared at least a one-way serotypic relationship with rotaviruses representative ofeach of the four established human serotypes (table 1). 4 -

5 Human Rotavirus Studies in Volunteers 875 Table 1. Cross-neutralization of human rotaviruses with hyperimmune guinea pig sera. Immunizing Antisera titers Guinea pig virus Wa DS-1 P ST-3 CJN 786 Wa 70,000 <100 (>700) 280 (250) <100 (>700) 3,400 (20.6) NIH* Wa 60, (120) 1,900 (32) 190 (320) 14,500 (4.1) 707 DS (240) 60,000 1,000 (60) <100 (>600) 15,000 (4.0) 708 DS (96) 24,000 1,100 (22) <100 (>240) 2,600 (9.2) 705 P 900 (92) <100 (>830) 83,000 <100 (>830) 2,000 (42) 706 P 900 (310) 270 (1,040) 280,000 <100 (>2,800) 4,700 (60) 98 ST-3 1,700 (410) <100 (>7,000) 4,300 (160) 700,000 9,500 (74) 100 ST (420) <100 (>4,000) 1,000 (400) 400, ,000 (2.9) 176 CJN 6,200 (6.1) 56 (680) 2,100 (18.1) 150 (250) 38, CJN 8,500 (4.4) 390 (105) 4,200 (8.8) 125 (300) 37,000 NOTE. Data are reciprocals of the serum dilution required to reduce plaque formation by 600;0 (homologous/heterologous ratios). * Reference antiserum from the National Institutes of Health. That is, when the CJN virus was compared with each of the other four human rotaviruses, homologous/heterologous ratios of <20, a ratio typically used to indicate serotypic relatedness [21-23], were found with at least one guinea pig antisera. No serotypic relatedness (homologous/heterologus ratios, >200) was observed when antisera to animal rotaviruses (NCDV [bovine] and OSU [porcine]), which are serotypically distinct from each other and from the human prototype strains [13], were used to neutralize the CJN virus (data not shown). Other investigators have found human [24] and animal [25] rotaviruses that are serotypically related to strains belonging to two serotypes, but relatedness to more than two serotypes has not been reported. For determination of whether the hyperimmune sera to the prototype strains used in this experiment had unusually broad reactivity, their plaque neutralization titers were measured against eight additional human rotaviruses isolated in this laboratory. Two isolates (nos. 66 and 70) were related to two of the four prototype strains in this one-way test, but the other six were related to only one (nos. 5, 21, 62, and 69) or none (nos. 1 and 4) of the prototype strains (table 2). Thus these antisera did not appear to have unusually broad reactivity. In addition, preimmunization antisera obtained from these guinea pigs contained no detectable antibody to rotavirus, as determined by an ELISA. This finding indicates that the broad neutralizing capabilities of these antisera toward the CJN virus were not due to an anamnestic response. Determination ofinfectious dose in human subjects. Subjects used for this study were first screened for serum neutralizing antibody to the "reassortant" CJN virus, and volunteers with titers of<30 were included. When these sera were retested with the plaque-purified CJN virus, however, titers Table 2. Homologous/heterologous ratios of neutralization titers of antisera to prototype strains of human rotavirus tested against eight human rotavirus isolates Homologous/heterologous ratios with isolate Immunizing Guinea pig virus Wa > NIH* Wa NO NO DS-I > ItO 708 DS >240 > > P 120 >830 > > > P 400 >2,800 >1,400 1,470 > >1, ST >7, , too ST >4, , NOTE. ND = not determined. * Reference antiserum from the National Institutes of Health.

6 876 Ward et al. ranged from <2 to 1,600, with a GMT (± SD) of 97.7 ± 6.8. After ingestion of the unpassaged, filtered, safety-tested preparation of this virus, subjects were monitored for clinical illness, shedding of virus, and serum antibody response (table 3). Twentytwo subjects shed detectable amounts of virus, and 21 of these also experienced a significant rise in titer of serum neutralizing antibody to CJN virus (i.e., greater than twofold). However, eight subjects who had significant rises in antibody titer did not shed detectable virus. Seventeen of the 30 subjects who seroconverted, shed virus, or both were also symptomatic for rotavirus illness. Three additional subjects (nos. 161,238, and 289) experienced clinical illness without evidence of shedding of rotavirus or seroconversion. Although their illnesses may have been due to rotavirus, they are not considered as such for statistical consideration because infection was not confirmed by the more specific and objective laboratory methods. Thirteen subjects experienced temporary elevations in serum transaminase activity above "normal" levels during this study, i.e., serum glutamic oxaloacetic or pyruvic transaminase activity of >50 IV/liter. However, the increases were not significantly greater or more common among infected than uninfected subjects. For example, seven of 30 infected subjects experienced elevated enzyme levels along with six of 32 uninfected subjects, two of whom received placebo. The degree of elevation was also not significantly different between infected and uninfected subjects. Shedding of virus was first detected between days 3 and 7 after inoculation and in four of 22 subjects continued until at least days No shedding was detected on days The time of illness overlapped with the time of shedding of virus in sub- Table 3. antibody. jects in whom both were detected. The 15 subjects who shed the greatest amount ofvirus, as determined by an ELISA, experienced higher rises in titer of serum neutralizing antibody than did the 15 subjects who shed lesser or undetected amounts of virus but also seroconverted (P =.08 by Kruskel-Wallis test [19]). The probability of illness in infected subjects was, however, unrelated to the quantity ofvirus shed by these subjects. Probit analysis of the data [20] was used to predict the relation between the infectivity of the challenge virus in cell culture and in humans (figure 2). Because ofthe distribution of values, fiducial limits were very wide. The results of this analysis did suggest, however, that the human infectious dose was 1"\.110 ffu, and 1 ffu should infect 1"\ of susceptible adult subjects. Thus infectivity in cell culture appeared to be comparable to that found in humans. Although only 17 of the 30 subjects shown to be infected experienced illness, the probability of illness in infected persons was not related to dose. It appeared therefore that the dose required to cause infection was comparable to that needed to produce illness. Seroconversion to heterologous rotaviruses. For determination of the breadth and magnitude of the heterotypic serological response, rises in titers of serum neutralizing antibody to viruses representative of the four serotypes of human rotavirus were measured in 16 infected subjects (table 4). Increases in titer of serum neutralizing antibody were observed to all four viruses in each of the 16 subjects, and the increase was less than twofold in only three instances (subjects 174 and 190 versus ST-3 and subject 183 versus P). When the magnitudes ofthe rises in antibody titer to these four viruses were ranked, average Dose response to the CJN strain of human rotavirus given to subjects with low titers of serum neutralizing Illness* Shedding of Multiple of rise Dose (ffu) Subject a b c d e g h virus (days) in antibody titer Infection 9 x , x , , continued

7 Human Rotavirus Studies in Volunteers 877 Table 3 (Continued). IlIness* Shedding of Multiple of rise Dose (ffu) Subject a b c d e f g h virus (days) in antibody titer Infection 9 X > , x x x * Illness was associated with one or more of the following symptoms: a = diarrhea; b = cramps; c = nausea; d = fever (temperature, ~100 F); e = headache; f = malaise; g = chills; and h = vomiting.

8 878 Ward et al. 0.1 O.O~--:=;;~_~~_~ ~_... ~_~ ~_~_~ -4 LOg l O 2 (dose) increases were greatest against Wa~ followed by ST 3, DS-l~ and P~ in descending order. However, almost the opposite ranking was observed when the final titers of neutralizing antibody were compared ( P > Wa > DS-l >ST-3).Therefore infection ofthese 16 subjects with the CJN strain of rotavirus consistently caused an increase in titer of serum neutralizing antibody to rotaviruses representative of each of the four established human serotypes, and the magnitude of this increase was similar against all four. Discussion The public health significance of human exposure to small quantities of infectious rotaviruses has not been previously assessed. The results of this study, performed with an unpassaged strain of human rotavirus, indicated that the dose required to infect susceptible adults was similar to that needed to cause infection in cell cultures. Although not all infected subjects became ill, the percentage infected who ex- Table 4. Titers of neutralizing antibody to different strains of human rotavirus in sera obtained before (day 0) and after (days 26-28) administration of CJN virus to 16 infected subjects. GMT Multiple of rise in Viral strain Day 0 Days antibody titer CJN , Wa , DS-l p , ST J Figure 2. Probit analysis ofthe probability of infection with CJN virus in human subjects as a function of dose (ffu). perienced illness was unrelated to dose. Therefore the dose of the virus required to cause human infection can also cause illness. As judged by the low dose found to cause bothinfection and illness in this study, the spread of rotavirus disease could readily occur through exposure to very small amounts of infectious virus, even in adults. Subjects were selected for this study based on their low «30) titers of serum neutralizing antibody to the tissue culture-adapted challenge virus. The virus used in this selection procedure, however, was a reassortant between CJN and a second rotavirus present in the original stool but not in the filtered stool prepartion used to challenge volunteers. Preinoculation titers of serum neutralizing antibody to the "reassortant" virus were very different from those to the tissue culture-adapted CJN virus itself. The range against triply plaque-purified CJN virus was <2 to 1,600, withagmt(± SD)of97.7 ± 6.8. Thus many subjects had relatively high titers of serum neutralizing antibody to the cultured CJN strain. High antibody titers, however, had no significant effect on the probability of either infection or illness in subjects given an infectious doseofrotavi rus, i.e., ~9 ffu, as determined by Student's t test. The 29 subjects in this category who became infected had a GMT (± SD) of108.9 ± 6.7, whereas the seven subjects given an infectious dose who did not become detectablyinfected hada GMT (± SD) of198.3 ± 2.3 to the CJN virus at the time of inoculation. Furthermore, eight of the nine subjects with the highest preinoculationtiters (GMT ± SD~ ± 2.3) became infected, and five of these experienced

9 Human Rotavirus Studies in Volunteers 879 illness. Therefore these concentrations ofserum neutralizing antibody to the challenge virus had no significant protective effect on subjects in this study. The uniqueness of the human rotavirus used in this study was determined by electrophoretic and serotypic analysis in comparison with other rotavirus strains. The electrophoretic pattern of RNA from the CJN virus was distinct from that of all other rotavirus strains tested. The more interesting feature of this virus, however, was revealed in crossneutralization experiments with rotaviruses representative of the four established human serotypes. At least a one-way serotypic relationship was observed between CJN virus and each of these four viruses. This broad serotypic relatedness was not observed when antisera to the four prototype strains was used to neutralize each other or additional human rotaviruses isolated and cultured in this laboratory. Thus the serotype ofcjn is unclear because it shares neutralization epitopes with all four established human serotypes. The practical significance ofserotype designation as it applies to rotaviruses remains to be determined. Others have shown that infection with some animal and human rotavirus strains can cause rises in titers of serum neutralizing antibody to viruses assigned to heterologous serotypes [8, 26-28]. Taniguchi et al. [29] reported that two of 11 hybridomas generated after immunization with rotavirus secreted neutralizing antibody that was commonly reactive to the three serotypes of human rotavirus tested. This finding indicated that different rotavirus serotypes share antigenic sites involved in neutralization, a suggestion that has important implications in vaccine development. Our results have shown (table 4) that adults infected with the CJN virus had similar rises in titer of serum neutralizing antibody to representatives of the four established serotypes of rotaviruse This observation is further evidence that the CJN virus shares neutralization epitopes with each of these four viruses. The suggestion that heterotypic protection may result after rotavirus immunization of both animals and humans [30, 31] leads to further questions regarding the practical significance of classifying rotaviruses as distinct serotypes. Based on the high rate of infection and illness found in subjects inoculated during this study, many cases of adult gastroenteritis may be due to rotavifus infection. Because the dose required to cause infection and illness in adults was very small, exposure of susceptible adults to an infectious dose must be a common occurrence. These results, coupled with with lack of protection associated with the presence of relatively high titers of serum neutralizing antibody to the challenge virus, indicate that multiple rotavirus infections occur during a lifetime. Lack of extensive evidence supporting a high incidence of rotavirus illness in adults has several possible explanations. For example, the infectivity of rotavirus found in this study may have been augmented over that occurring in nature because of the consumptionof bicarbonate. Food, however, may have a similar effect. Another explanation for the low reported incidence of rotavirus disease in adults is differences in disease manifestation in infected adults compared with children. This factor may be the result of a difference in the susceptibility of adult enterocytes to rotavirus infection, as observed in studies with animals [32, 33] or a partial protection provided by prior rotavirus infections. It is also possible that adult disease due to rotavirus is quite common but the agent is not identified because the amount of virus shed falls below the limit of detection by present assay methods. Our results indicate that adults can experience rotavirus illness, as documented by seroconversion, without shedding sufficient virus to be detected by a sensitive ELISA (see subjects 117,243, 212, and 128 in table 3). Consideration should be given to these results in subsequent epidemiological studies. References I. Kapikian AZ, Chanock RM. Rotaviruses. In: Fields BN, Knipe OM, Chanock RM, Melnick JL, Roizman B, Shope RE, eds. Virology, New York: Raven Press, 1985: Flewett TH. Rotavirus in the home and hospital nursery. Br Med J 1983;287: Ward RL, Knowlton DR, Pierce MJ. Efficiency of human rotavirus propagation in cell culture. J Clin Micribiol 1984;19: Moe K. Shirley JA. The effects of relative humidity and temperature on the survival of human rotavirus in faeces. Arch Viro1 1982;72: Hung T, Chen G, Wang C, Yao H, Fang Z, Chao T, Chou Z, YeW, Chang X, Den S, Liong X, Chang W. Waterborne outbreak of rotavirus diarrhoea in adults in China caused by a novel rotavirus. Lancet 1984;1: Ward RL, Akin EW. Minimun infective dose of animal viruses. CRC Critical Reviews in Environmental Control 1984;14: Schiff GM, Stefanovic GM, Young EC, Sander OS, Pennekamp JK, Ward RL. Studies of echovirus-12 in volunteers: determination of minimal infectious dose and the effect of previous infection on infectious dose. J Infect Dis 1984;150:858-66

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