Development of Neutralizing Antibodies and Group A Common Antibodies against Natural Infections with Human Rotavirus

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1 JOURNAL OF CLINICAL MICROBIOLOGY, Aug 1988, p /88/ $02/0 Copyright C 1988, American Society for Microbiology Vol 26, No 8 Development of Neutralizing Antibodies and Group A Common Antibodies against Natural Infections with Human Rotavirus BO-JIAN ZHENG,"12t SHOU-XIN HAN,' YONG-KAI YAN,' XI-RUO LIANG,3 GUI-ZHANG MA,3 YING YANG,3 AND M H NG2* Department of Microbiology and Immunology, Sun Yat-Sen University of Medical Sciences,' and Department of Microbiology and Immunology, Guangzhou Medical College,3 Guangzhou, People's Republic of China, and Department of Microbiology, Faculty of Medicine, University of Hong Kong, Hong Kong2 Received 1 February 1988/Accepted 3 May 1988 We determined the levels of group A common and neutralizing antibodies against human rotavirus in paired serum specimens obtained from 38 infants within 12 days of the onset of diarrhea Thirty of the infants excreted rotavirus in stools, and eight did not Nine patients (30%) with rotavirus diarrhea and seven patients (88%) with diarrhea due to other causes had detectable levels (1:80) of immunoglobulin (IgG) common antibodies in acute-phase sera All the patients with rotavirus diarrhea showed at least fourfold rises in titers of IgG or IgM common antibodies or both, while only two control patients showed significant rises in either IgG or IgM common antibodies in their convalescent-phase sera Of the 19 patients excreting "short" electropherotypes of rotavirus, 18 showed at least fourfold rises in titers of neutralizing antibodies against serotype 2 human rotavirus but not against serotype 1, 3, or 4 Nine of the ten patients excreting "long" electropherotypes showed significant rises in neutralizing antibodies against serotype 3, and the other patient showed a significant rise in neutralizing antibodies against serotype 1 One patient excreted long and short electropherotypes simultaneously, and he also showed a significant rise in neutralizing antibodies against serotype 2 and 3 viruses The control patients with diarrhea did not show significant changes in titers of antibodies against any of the serotypes These results demonstrated that the neutralizing antibody response within 2 weeks after clinical onset is specific for the infecting serotype of rotavirus Human rotavirus (HRV) is an important cause of diarrhea iri infants and young children worldwide; it is responsible annually for an estimated 5,0,0 cases of infantile diarrhea in developing countries (7) The disease shows a marked seasonal distribution, being most prevalent during the winter, and the genetic makeup of the virus population in a community is varied and constantly changing (12, 24, 28) The antigenic properties of the virus are likewise complex and varied There are at least six known serotypes of HRV (1, 5, 16, 25) Protection against HRV infections is not well understood, although studies in animals have demonstrated that antibodies present in the lumen of the small intestine appear to be the prime mediators of protection against rotavirus infection (17, 23) Recurrent rotavirus infection has been documented, and the infection can occur despite the presence of serum antibodies against group A common or subgroup-specific rotavirus antigens (13, 22) The antigenic specificity of neutralizing antibodies is believed to be principally directed against the viral proteins VP3 and VP7 on the outer viral capsid, and these viral proteins are encoded by segments 4 and 9 or 8 of the viral genome (11, 15, 18) Apart from the serotype-specific neutralizing antibodies, natural infection may elicit the production of neutralizing antibodies against other serotypes of HRV (3, 4, 9, 19) To better study the antibody response to natural rotavirus infection, we obtained paired serum specimens from 30 patients with diarrhea caused by rotavirus and 8 patients with diarrhea due to other causes The sera were tested for common antibodies and for neutralizing antibodies * Corresponding author t Present address: Department of Microbiology, Faculty of Medicine, University of Hong Kong, Hong Kong 1506 (This work was conducted in partial fulfilment of the PhD requirements for B-JZ at the University of Hong Kong) MATERIALS AND METHODS Patients Stool specimens were obtained from infants with diarrhea on the day of admission to Red Cross Hospital, Guangzhou, People's Republic of China, between October 1984 and January 1985 The specimens were tested by enzyme-linked immunosorbent assay for the presence of rotavirus, as described by Yan and Zheng (28) Thirty patients who gave positive results for rotavirus and eight patients who gave negative results were selected for further study Daily stool specimens were obtained from the two groups of patients Paired serum specimens were also obtained, one specimen usually on the day of admission and the other after the symptoms had subsided Virological investigations The stool specimens were tested by enzyme-linked immunosorbent assay for the presence of rotavirus The results were confirmed by polyacrylamide gel electrophoresis of the viral RNA extracted from the stool specimens by the method of Herring et al (10) The patterns of viral RNA were analyzed and classified, as previously described by Tam et al (24) Serology The serum specimens were tested for immunoglobulin G (IgG) and IgM antibodies against the group A rotavirus common antigens (common antibodies) and for neutralizing antibodies against the four serotypes of the prototype HRV, ie, Wa (serotype 1), S2 (serotype 2), Yo (serotype 3), and Hochi (serotype 4) Rotavirus common antibody was assayed by the method of Bishop et al (2), except that bovine strain NCDV propagated in MA104 cells was used instead of simian strain SA11 The cells were harvested 1 day after infection with NCDV They were frozen and thawed twice, sonicated six

2 VOL 26, 1988 ANTIBODY RESPONSE TO HRV INFECTION 1507 TABLE 1 Study patients with diarrhea due to rotavirus or other causes Cause of No of No of Age (mo) Mean duration (days ± SD) of: diarrhea patients males/females Range Mean ± SD Diarrhea HRV shedding Rotavirus 30 22/ Other 8 4/ ± ± 16 None a The patients with diarrhea were admitted to Red Cross Hospital, Guangzhou, between October 1984 and January 1985 times for 10 s each time at 20 khz, and centrifuged at 10,0 rpm for 20 min at 4 C The supernatant was homogenized with an equal volume of trichlorotrifluoroethane in a lowspeed blender and centrifuged at 10,0 rpm for 20 min at 4 C The aqueous phase was pooled and stored at -70 C until use The neutralizing antibodies were determined by the fluorescence foci neutralization assay described by Beards et al (1) Briefly, microdilution plates were seeded with approximately 2 x 104 MA104 cells per well and incubated for 24 h at 37 C The cultures were then washed in serum-free medium, and all subsequent operations were done in the absence of fetal bovine serum The cultures were challenged with about 2 fluorescence focus-forming units of the prototype viruses which had previously been mixed with and incubated for 1 h at 370C in an equal volume (01 ml) of serially diluted aliquots of sera from patients The stock virus preparations had been incubated for 05 h before use in Eagle minimum essential medium containing 20,±g of trypsin per ml After 20 h of incubation, the cultures were washed and the cells were fixed by being treated with ethanol at -20 C and then air dried To determine the number of infective foci, the cultures were reacted for 15 h at 37 C with a 1:1 diluted guinea pig hyperimmune serum produced against NCDV, washed three times, and then similarly reacted with 1:1 diluted antiguinea pig immunoglobulin-fluorescein isothiocyanate conjugate The cultures were washed, and the number of fluorescence foci was determined The neutralizing activity of the serum was indicated by the reduction in the number of fluorescence foci from that obtained with the virus control cultures which had not been treated with the sera from patients but which were otherwise similarly tested in parallel The neutralizing antibody titer was obtained by using linear regressions of the percent reduction in fluorescence foci on the log of the reciprocal serum dilution, and it was defined as the reciprocal of the serum dilution which caused 50% reduction in fluorescence foci The fluorescence foci neutralization assay for a serotype was done in duplicate Pairs of acute- and convalescentphase serum specimens were tested concurrently A panel of standard sera obtained from laboratory personnel was included on each occasion The variation in neutralization titers obtained on separate occasions was less than twofold Materials The hyperimmune guinea pig serum was produced by four intramuscular inoculations at 2-week intervals with the single-capsid particles of NCDV mixed in equal volumes of Freund incomplete adjuvent The single-capsid particle was purified by banding at 138 mg/ml after centrifugation in a CsCl gradient (20) MA104 cells derived from rhesus monkeys were passaged in Eagle minimum essential medium containing 10% fetal bovine serum The four serotypes of prototype HRV, ie, Wa, S2, Yo, and Hochi, and the bovine virus, NCDV, were propagated in MA104 cultures in the presence of trypsin, as described by Sato et al (21) RESULTS Patients We studied 38 infants admitted with diarrhea to Red Cross Hospital in Guangzhou (Table 1) Repeated stool specimens obtained from 30 of them gave positive enzymelinked immunosorbent assay results for rotavirus, and the infants continued to shed the virus for an average of 63 days The remaining eight patients gave negative results for rotavirus The two groups of patients were otherwise comparable with respect to age and duration of diarrhea Virological findings As shown in Table 2, six electropherotypes, all of which corresponded to the electropherotypes which were prevalent in the city at that time, were identified from 29 patients with rotavirus diarrhea The frequencies of isolation of these electropherotypes were also comparable to the relative prevalences of their isolation in the city for the period of the present study Two of these electropherotypes were simultaneously isolated from the one remaining patient, and both of these electropherotypes were cocirculating in the city during that time The six electropherotypes of the rotavirus isolates are shown in Fig 1 Three of them showed a "'short" pattern due to slow migration of segments 10 and 11 of the viral genome They were designated IA4, IA5, and IB1 ("TI" refers to the short pattern, as described by Tam et al [24]) These electropherotypes are usually obtained with serotype 2 HRV (22) By coelectrophoresis of IA5 with IA4 or IB1, it was shown that the three isolates were indeed different electropherotypes The other three isolates showed a "long" pattern, and they were designated IIA4, IIB3, and IIE1 ("II" refers to the long pattern) The differences between these electropherotypes were similarly confirmed by coelectrophoresis of IIA4 with IIB3 or IIE1 Group A HRV common antibody response Paired serum specimens were obtained from the 38 patients The first specimens were taken on admission from day 1 to day 6 after the onset of disease, and the second specimens were taken after 5 to 12 days, when the symptoms had subsided They were titrated for neutralizing antibodies against serotype 1 to 4 prototype HRV, Wa, S2, Yo, and Hochi, and for antibodies against group A common antigens, as described in Materials and Methods TABLE 2 HRV electropherotypes excreted by patients and prevalence in Guangzhou at the time of the study Electropherotype(s) Noof patiets excretingthevirus the virus Prevalence in communityn (222 isolates) IIE IIA IIB IA IA IB IIA4 and IA a Classification of electropherotypes is according to Tam et al (24) I, Short pattern; II, long pattern

3 1508 ZHENG ET AL J CLIN MICROBIOL s*sivsb~~~ 11} ~~~1Ii 4 C 1:`7; TABLE 4 and I 11A4 A 1 1IA I131 11AA 1 kdit No of FIG 1 Identification of electropherotypes isolated from 30 patients with diarrhea Six electropherotypes were isolated The differences between them were confirmed by coelectrophoresis of the three isolates with the long pattern (II) and the three isolates with the short pattern (t) One patient was concurrently infected with two electropherotypes, ie, IA5 and IIA4 The 11 segments of the viral genome are identified numerically Of the patients with rotavirus diarrhea, 70% (21 of 30) did not show detectable levels (<1: 80) of IgG common antibody in acute-phase sera, whereas acute-phase sera from the remaining 9 patients (30%) with rotavirus diarrhea had levels of IgG common antibodies of 21: 160 By contrast, seven of the eight control patients with diarrhea might have had prior infection with rotavirus; the titers of IgG common antibodies in their acute-phase sera exceeded 1:160 The titers of IgG and IgM common antibodies obtained with the convalescent-phase sera were compared with the respective values obtained concurrently with the corresponding acute-phase sera (Table 3) All 30 of the patients with rotavirus diarrhea showed significantly increased titers of either one or both of these antibodies in the convalescentphase sera Of the eight patients who did not yield rotavirus in their stool specimens, six also did not show significant rises in the titer of either of the antibodies One of the two remaining control patients with diarrhea showed a fourfold rise in the titer of IgM common antibodies only, and the other showed a fourfold rise in IgG common antibodies; neither of them also showed significantly increased titers of neutralizing antibodies (see Fig 2C) Neutralizing antibody response Fig 2 compares the titers of neutralizing antibodies against the four serotypes of prototype HRV obtained with the acute- and convalescentphase sera from 28 patients with rotavirus diarrhea and 8 control patients with diarrhea Of the former group of Development of rotavirus common antibodies in patients with diarrhea due to rotavirus or other causes TABLE 3 Log-2 increase of common antibodiesa antibodiesofno of patients with diarrhea due to: IgG IgM Rotavirus Other causes 2: < < <4 <4 0 6 a A significant antibody response was indicated when the antibody titer obtained with the convalescent-phase serum exceeded that obtained with the acute-phase serum by fourfold or more Serotype-specific development of HRV neutralizing antibodies Log-2 increase in titers of neutralizing antibodies against patients Electropherotype(s) HRV serotype: l IIE IIA4 or IIB IAS, IA4, or IB IIA4 and IA None a The development of antibodies is indicated by a log-2 increase in the titer of antibodies obtained with the convalescent-phase sera from the titers obtained with the corresponding acute-phase sera Significant increases in antibody titers are indicated in boldface type patients, 18 showed significant rises of more than fourfold in the titers of neutralizing antibodies against serotype 2 of the prototype HRV, S2, while the titers of neutralizing antibodies against serotype 1, 3, and 4 prototype rotaviruses did not change significantly (Fig 2A) An additional patient showed a marginal increase of 32-fold in neutralizing antibodies against serotype 2 virus but no increase in antibodies against the other serotypes The convalescent-phase serum from this infant was obtained on day 6 after the clinical onset of disease, and this may not have allowed sufficient time for antibody development (see Fig 3 and 4) It was noted that all 19 of these patients excreted the short electropherotypes, which are usually obtained with subgroup 1, serotype 2 HRV (25) For these patients, therefore, the results of virus isolation are consistent with the serological finding of specific neutralizing antibody responses to serotype 2 virus The specificity of the neutralizing antibody response was further evidenced by the results obtained from nine patients, eight of whom yielded IIA4 virus and one of whom yielded IIB3 virus in repeated stool specimens Fig 2B shows that all these patients had rises of between 64- and 224-fold in the titer of neutralizing antibodies against serotype 3 of the virus, while the titers of neutralizing antibodies against the other serotypes did not change significantly By contrast, Fig 2C shows that the sera obtained from the eight control patients with diarrhea did not show significant changes in titers of neutralizing antibodies against any of the four serotypes The above-described results and those obtained with two other patients with rotavirus diarrhea are summarized in Table 4 One of the latter patients yielded IIE1 virus from his stool specimens, and he showed a 65-fold rise in titer of neutralizing antibodies against the serotype 1 HRV strain, Wa The other patient simultaneously yielded two electropherotypes (IIA4 and IAS), and the infection also elicited 72- and 103-fold rises in titers of neutralizing antibodies against serotype 2 and 3 viruses, respectively Thus, it was clear that those isolates with the short electrophoretic pattern, ie, IA4, IA5, and IB3, are antigenically related to serotype 2 prototype virus, such that infection with these electropherotypes elicited specific neutralizing antibody responses against that virus On the other hand, the electropherotypes with the long electrophoretic patterns might be related either to serotype 1, as is IIE1, or to serotype 3, as are IIB3 and IIA4 To date, we have not encountered infection with serotype 4 viruses, however Antibody response related to virus shedding and clinical course of diarrhea Fig 3 shows that the patients with

4 VOL 26, 1988 ANTIBODY RESPONSE TO HRV INFECTION 1509 (A) (B) Setype i is o O O Neutralzkng tr (acute sra) FIG 2 Occurrence of HRV neutralizing antibodies in acute- and convalescent-phase sera from patients with diarrhea One group of patients excreted electropherotype IA5, IA4, or IB1 (A); one group excreted IIA4 or IIB3 (B); and the other group had diarrhea due to other causes and did not excrete the virus (C) 6' 4 Neutralzing t#ter (aut (c) 6' 4' sera) Sertype 3 Fig 4 compares the development of neutralizing antibodies against the serotype of HRV believed to have caused the diarrhea with virus shedding and clinical course of the disease The results show that rising titers of neutralizing antibodies coincided with the time of symptomatic recovery from the disease and the time when virus shedding subsided S e N 4 Z / Sotype2 2 4 O ' o 6' 4 2 DISCUSSION ( ~ We have described the development of neutralizing anti- 0 1 Î0 3S0 4 bodies and antibodies against the antigen of A rotavirus in a group of 30 infants with confirmed rotavirus Serype 4 diarrhea As a control, we also studied the antibody responses in eight patients with diarrhea due to other causes, the diagnoses for six of whom were supported by negative virological and serological findings One of the remaining control patients showed a fourfold rise in IgM common antibodies, and the other showed a fourfold rise in IgG ^common antibodies Neither of the patients, however,,r yielded rotavirus in his stool specimens, nor did they show significant increases in the level of neutralizing antibodies Neutazng titerr(aoute ) Therefore, it was considered unlikely that their diseases were caused by rotavirus Both groups of patients were otherwise comparable with respect to age and duration of the disease rotavirus diarrhea mounted vigorc>us antibody responses to Neutralizing antibodies were titrated by the fluorescence the infection, and this was so wit] h respect to IgM and IgG foci neutralization assay (27), which is more convenient to common antibodies and the neutrralizing antibodies against perform than the conventional assay by plaque reduction; the HRV serotypes believed to have caused the disease the antigenic specificities of the two methods have been Rising titers of these antibodies uvere evidenced between 5 shown to be similar (6, 8) We showed that the neutralizing and 6 days after the onset of diseaçse There was no apparent antibody response to natural rotavirus infection is serotype difference between the production )n of common antibodies specific Thus, 29 of the 30 patients with rotavirus diarrhea and the production of neutralizing antibodies developed neutralizing antibodies against single serotypes of

5 1510 ZHENG ET AL Y? _ q> M -an Z 5 O O _ ' Days after clinical onset Number of serum sample FIG 3 Development of rotavirus antibodies Serum specimens were obtained from the indicated numbers of patients with rotavirus diarrhea on different days after the onset of disease and tested for rotavirus antibodies The geometric mean titer (GMT) values for the neutralizing antibodies against the serotypes of HRV that caused the disease and IgG and IgM common antibodies were calculated from these results J CLIN MICROBIOL HRV These patients also yielded single electropherotypes in repeated stool specimens The remaining one patient was concurrently infected with two electropherotypes, and this patient also developed neutralizing antibodies against both serotype 2 and 3 prototype viruses None of the eight control patients with diarrhea showed significantly increased titers of neutralizing antibodies, however Nineteen patients developed neutralizing antibodies against serotype 2 virus The viruses isolated from them, ie, IA4, IA5, and IB1, yielded a short electrophoretic pattern, as did the serotype 2 virus, S2 It was shown previously that the short electrophoretic pattern is usually obtained with subgroup 1, serotype 2 HRV (25) Thus, for these patients, the virological and serological findings were in agreement, confirming serotype 2 HRV as the cause of the infection Apart from the aforementioned, electropherotyping results do not seem to be related to the functional or antigenic properties of rotavirus (see review by Estes et al [8]) Indeed, infection with either IIA4 or IIB4 elicited in nine patients the development of neutralizing antibodies against the serotype 3 prototype virus, Yo, while in another patient, infection with a similar virus, IIE1, elicited the development of neutralizing antibodies against the serotype 1 virus, Wa There are at least six known serotypes of human rotavirus (1, 5, 16, 25) However, serotype 4 virus was not encountered in the present study, and we did not specifically study antibody response to serotype 5 and 6 viruses Kapikian et al (14) showed that vaccination with HRV _ 14 13W 12 - L > 8 - cc I _Osoo~ E 4* 3 2-1* O, 0,, 01 0 H,, Days after dinicai onset Number of sseum sampls I R 24 g 22? d z a 8 *MI 6 -S 4 3M 2 E FIG 4 Development of neutralizing antibodies in the course of diarrhea Thirty patients were monitored clinically and for shedding of rotavirus The geometric mean titers (GMT) of neutralizing antibodies against HRV serotypes that caused the disease were calculated with the results obtained with the indicated number of serum specimens taken from these patients on the days shown :1 r-4

6 VOL 26, 1988 elicited in adult volunteers the production of neutralizing antibodies against both the vaccine strain and the other serotypes In that instance, the heterotypic antibodies produced might have been due in part to anamnestic responses to previous infections with the virus However, heterotypic responses were also reported for infants, many of whom probably had primary infections with the virus (3, 4, 9, 19) There are two possible explanations for the discrepancies between these and the present findings Firstly, the development of heterotypic antibodies may occur later than that of the serotype-specific antibodies Since all but one of the serum specimens in the present study were obtained within 10 days of the onset of disease, there may not have been sufficient time for the levels of heterotypic antibodies to increase Although none of the present group of patients with rotavirus diarrhea showed significant development of heterotypic antibodies, it was noted nevertheless that the average increases in the levels of heterotypic antibodies were consistently greater for the group of patients with rotavirus diarrhea than for the group of control patients with diarrhea (Table 4) Secondly, the production of heterotypic antibodies appears to depend on the serotype that caused the infection Thus, Gerna et al (9) showed that infection with serotype 3 virus additionally elicited heterotypic antibodies against serotype 1, while infection with serotype 1 or 2 elicited neutralizing antibodies against the infecting serotype only Extending the observations by these investigators (9), the serotype 2 viruses encountered in the present study comprised three different electropherotypes, all of which elicited production of neutralizing antibodies specific for that serotype only However, Chiba et al (3) showed that a serotype 3 virus that caused an outbreak additionally elicited heterotypic antibodies against serotype 1 and 4 viruses, while the two electropherotypes comprising the serotype 3 viruses cocirculating in the community during the present study elicited only serotype-specific neutralizing antibody responses We did not follow up these patients to determine whether they later developed heterotypic antibodies, however Previously, we and others showed that the rotavirus population in a community is genetically diverse; different electropherotypes often cocirculate in a community, and the virus population of the community changes with time The present findings extend the earlier observations, showing that different serotypes of HRV also cocirculate in a community and that the prevalent serotypes may change with time Furthermore, it seems that natural infection with a virus may lower the risk of individuals to subsequent symptomatic infection with the different serotypes cocirculating in the community Although protection is primarily due to antibodies in the lumen of the intestine (17, 23), the protective effect may also be ascribed, in part, to the presence of serum antibodies against the virus The latter contention is not inconsistent with the results of several recent vaccine trials (26, 27) and the development of heterotypic antibodies after natural infection with HRV (3, 4, 9, 19) However, it was noted that nine patients had elevated titers of HRV antibodies at the onset of HRV diarrhea For three of these patients who were 6 months old, the antibodies might have been of maternal origin The other six patients were older than 6 months of age, and the elevated HRV antibody titers might have been due to previous infections Therefore, it symptomatic reinfection with HRV despite an seems that elevated level of serum antibodies is not an uncommon occurrence ANTIBODY RESPONSE TO HRV INFECTION 1511 ACKNOWLEDGMENT This work was partly supported by a grant from the Croucher Foundation LITERATURE CITED 1 Beards, G M, J N Pilford, M E Thouless, and T H Flewett 1980 Rotavirus serotypes by serum neutralisation J Med Virol 5: Bishop, R F, E Cipriani, J S Lund, G L Barnes, and C S Hosking 1984 Estimation of rotavirus immunoglobulin G antibodies in human serum samples by enzyme-linked immunosorbent assay: expression of results as units derived from a standard curve J Clin Microbiol 19: Chiba, S, S Nakata, T Urasawa, S Urasawa, T Yokoyama, Y Morita, K Taniguchi, and T Nakao 1986 Protective effect of naturally acquired homotypic and heterotypic rotavirus antibodies Lancet ii: Clark, H F, K T Dolan, P Horton-Slight, J Palmer, and S A Plotkin 1985 Diverse serologic response to rotavirus infection of infants in a single epidemic Pediatr Infect Dis 4: Clark, H F, Y Hoshino, L M Bell, J Groff, G Hess, P Bachman, and P A Offit 1987 Rotavirus isolate WI61 representing a presumptive new human serotype J Clin Microbiol 25: Cukor, G, and N R Blacklow 1984 Human viral gastroenteritis Microbiol Rev 48: Dimitrov, D H, M K Estes, S M Rangelova, L M Shindarov, J L Melnick, and D Y Graham 1983 Detection of antigenically distinct rotaviruses from infants Infect Immun 41: Estes, M K, E L Palmer, and J F Objesk 1983 Rotaviruses: a review Curr Top Microbiol Immunol 105: Gerna, G, M Battaglia, G Milenesi, N Passarani, E Percivalle, and E Cattaneo 1984 Serotyping of cell culture-adapted subgroup 2 human rotavirus strains by neutralization Infect Immun 43: Herring, A J, N F Inglis, C K Ojeh, D R Snodgrass, and J D Menzies 1982 Rapid diagnosis of rotavirus infection by direct detection of viral nucleic acid in silver-stained polyacrylamide gels J Clin Microbiol 16: Hoshino, Y, M M Sereno, K Midthun, J Flores, A Z Kapikian, and R M Chanock 1985 Independent segregation of two antigenic specificities (VP3 and VP7) involved in neutralization of rotavirus infectivity Proc Natl Acad Sci USA 82: Kapikian, A Z, H W Kim, R G Wyatt, W L Cline, J O Arrobio, C D Brandt, W J Rodriguez, D A Sack, R M Chanock, and R H Parrott 1976 Human reovirus-like agent as the major pathogen associated with winter gastroenteritis in hospitalized infants and young children N Engl J Med 244: 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 diarrhea and development of serologic tests Science 185: Kapikian, A Z, R G Wyatt, M M Levine, R H Yolken, D H Vankirk, R Dolin, H B Greenberg, and R M Chanock 1983 Oral administration of human rotavirus to volunteers: induction of illness and correlates of resistance J Infect Dis 147: Lopez, S, C F Arrias, J R Bell, J H Trauss, and R T Espsjo 1985 Primary structure of the cleavage site associated with trypsin enhancement of rotavirus SA11 infectivity Virology 144: Matsuno, S, A Hasegawa, A Mukoyama, and S Inouye 1985 A candidate for a new serotype of human rotavirus J Virol 54: McNulty, M S, J B McFerran, D G Bryson, E F Logan, and W L Curran 1976 Studies on rotavirus infection and diarrhoea in young calves Vet Rec 99: Offit, P A, and G Blavat 1986 Identification of the two rotavirus genes determining neutralization specificities J Virol 57:

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