Belonging to Different Genogroups - NCBI

JOURNAL OF VIROLOGY,

JUlY 1990, p. 3219-3225

Vol. 64, No. 7

0022-538X/901073219-07$02.00/0 Copyright C) 1990, American Society for Microbiology

Evidence for Natural Reassortants of Human Rotaviruses Belonging to Different Genogroups RICHARD L. WARD,'* OSAMU NAKAGOMI,2 DOUGLAS R. KNOWLTON,' MONICA M. McNEAL,1 TOYOKO NAKAGOMI,3 JOHN D. CLEMENS,4 DAVID A. SACK,S AND GILBERT M. SCHIFF' Division of Clinical Virology, James N. Gamble Institute of Medical Research, 2141 Auburn Avenue, Cincinnati, Ohio 45219'; Departments of Laboratory Medicine2 and Microbiology3, School of Medicine, Akita University, Akita 010, Japan; Center for Vaccine Development, School of Medicine, University of Maryland, Baltimore, Maryland 212014; and Department of International Health, School of Hygiene and Public Health, The Johns Hopkins University,

Baltimore, Maryland 212055 Received 22 February 1990/Accepted 6 April 1990

Of 335 rotavirus isolates associated with diarrheal disease in Bangladesh that were culture adapted and subsequently characterized for electropherotype, subgroup, and serotype, 9 had properties that suggested they may be natural reassortants between human rotaviruses belonging to different "genogroups." Two of these were examined in greater detail by RNA-RNA hybridization with prototype strains representative of each of the three proposed human rotavirus genogroups. One subgroup II isolate, 248, with a "long" electrophoretic pattern was neutralized by hyperimmune antisera to both serotype 2 and 4 strains. Consistent with these results, seven RNA segments of this isolate formed hybrids with human strains belonging to the Wa genogroup and four segments hybridized with strains belonging to the DS-1 genogroup. The second isolate examined, 456, belonged to subgroup II and had a long electrophoretic pattern but was found to be a serotype 2 strain. This isolate also appeared to be an intergenogroup reassortant because three of its segments formed hybrids with strains belonging to the Wa genogroup and eight hybridized with viruses of the DS-1 genogroup. On the basis of the relative migration rates of these RNA-RNA hybrids during gel electrophoresis, a suggested origin for each gene segment was proposed which was consistent with the results expected from electrophoretic, subgroup, and serotypic analyses. The genome of rotavirus is composed of 11 segments of double-stranded RNA which encode at least 11 distinct viral proteins (8). Six serotypes of human rotaviruses have been identified (7, 19, 26), and two outer capsid proteins, VP4 and VP7, have been found to be neutralization antigens involved in serotype determination (15, 17, 18, 21, 33, 38). These rotavirus proteins are encoded by gene segments 4 and either 7, 8, or 9 (15, 18, 21, 33, 44). The inner capsid protein VP6 is the group antigen, and two subgroups (I and II) of human rotavirus have been identified on the basis of antigenic differences in this protein (16, 39). Until recently, all serotype 2 and 8 strains were found to belong to subgroup I and serotype 1, 3, 4, and 9 strains were found to belong to subgroup 11 (3, 7, 13, 19, 22, 34, 37). In addition, subgroup I strains were found to have characteristic "short" electropherotypes associated with an inversion in the migration order of segments 10 and 11 (20, 24). Thus, at least 4 of the 11 segments of human rotaviruses were found to be genetically linked. The AU-1 strain isolated in 1982 was the first natural human rotavirus strain in which this linkage appeared to have been broken (23, 30). Since that time, a number of investigators have reported the isolation of human rotaviruses with properties similar to those of AU-1 (1, 4, 5, 14, 35, 36). These viruses belonged to subgroup I but were identified as serotype 3 and had "long" electrophoretic patterns, properties common to many animal rotavirus strains. Although it has not been definitively shown that these are animal strains that have infected humans, it has been found that they are molecularly distinct from the two groups of human rotaviruses previously described (9-11, 28, 29, 31, *

32). This conclusion was based on RNA-RNA hybridization studies in which it has been shown that human rotaviruses within a group were much more genetically related to each other than to human strains in other groups. Thus, three human rotavirus "genogroups" have been defined on the basis of genetic homology with prototype strains Wa (subgroup II, long electropherotype), DS-1 (subgroup I, short electropherotype), and AU-1 (subgroup I, long electropherotype) (28, 32). Coinfection of cells with two strains of rotavirus can result in the formation of virus progeny that have inherited segments from both parental strains. Such reassortants were readily formed after coinfection of cultured cells with subgroup II human rotavirus strains, and some of these reassortants were selected in preference to the parental viruses (42). Reassortants were also formed after in vitro coinfection with subgroup I and II human rotavirus strains (12, 40, 41), but their isolation rates were lower and they were unable to effectively compete with the parental strains during multiple cell culture passages (41). In agreement with these findings, a presumed natural reassortant between subgroup II strains was reported some years ago, but natural reassortants between subgroup I and II human rotaviruses were not positively identified (18). Very recently, however, several laboratories have reported finding human rotaviruses which have atypical associations between subgroup, serotype, and electropherotype (2, 4, 25, 27, 36). This led to the suggestion that they may be natural reassortants between human rotaviruses belonging to different genogroups. A study on the epidemiology of rotavirus disease in the Matlab region of Bangladesh during 1985-1986 was recently completed, and 335 human rotavirus isolates were culture adapted and characterized by serological and electrophoretic

Corresponding author. 3219

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WARD ET AL.

analyses (R. L. Ward, J. D. Clemens, D. A. Sack, F. Ahmed, and N. Huda, Abstr. Sixth Int. Conf. Comp. Appl. Virol. 1989, W9-9). Two of these isolates that displayed atypical subgroup, serotype, and electropherotype associations were further characterized by RNA-RNA hybridization with segments from prototype human rotaviruses belonging to the Wa, DS-1, and AU-1 genogroups. This analysis has provided strong evidence that both isolates are natural reassortants between human rotaviruses of different genogroups. MATERIALS AND METHODS Isolation of rotavirus strains 456 and 248. The two human rotavirus strains examined in this study were isolated from the stools of subjects with diarrheal disease during an investigation conducted in Bangladesh during 1985 and 1986. These isolates were culture adapted by two passages in primary African green monkey kidney cells and eight subsequent passages in MA-104 cells as previously described (43). The culture-adapted preparations were plaque picked three times, and stock cultures of each were stored in samples at -700C. Electropherotyping of viral RNA. Polyacrylamide gel electrophoresis of genomic RNA segments extracted from virus preparations was conducted as previously described (42). Subgroup and serotypic analysis of isolates. Subgroup determination of virus isolates was made by using subgroupspecific monoclonal antibodies (255/60/125/14 and 631/9/ 104/56), which were generously provided by H. B. Greenberg. The analysis was conducted by using an enzymelinked immunosorbent assay as follows. Wells of microdilution plates were coated with rabbit anti-human rotavirus antibody (1:50 dilution; Dakopatts A/S, Copenhagen, Denmark). After overnight incubation at 40C, wells were washed and EDTA-treated virus diluted 1:1 in phosphate-buffered saline containing 5% nonfat dry milk (P-NDM) was added and left for 1 h at room temperature. Wells were washed, and subgroup-specific monoclonal antibodies produced in cell culture and diluted 1:20 in P-NDM were added and left for 30 min at room temperature. Wells were again washed, and horseradish peroxidase-conjugated rabbit anti-mouse immunoglobulin G (1:200 in P-NDM; Dakopatts A/S) was added and left for 30 min. Finally, wells were washed and incubated for 15 min with substrate (H202) and indicator (orthophenylenediamine). The reaction was stopped with 1 M H2SO4, and color development was determined by spectrophotometry. Serotypic analysis of virus isolates was made by using sera of guinea pigs hyperimmunized with purified preparations of Wa, DS-1, P, or ST-3 rotavirus, representative of serotypes 1 through 4, respectively. Antibody titers to each virus were determined by a focus reduction neutralization assay (4a). The lowest dilution (cutoff value) used was 1,000 for each antiserum. Titers are expressed as the reciprocal of the dilution required to reduce the number of focus-forming units by 60%. Preparation of dsRNA for hybridization analysis. Genomic double-stranded RNA (dsRNA) was extracted with phenolchloroform from partially purified virions which were prepared from infected MA-104 cells by pelleting at 40,000 rpm for 1.5 h in a Hitachi RP42 rotor and then by sedimentation through 30% (wt/vol) sucrose at 38,000 rpm for 2 h in a Hitachi RPS40T rotor. Preparation of ssRNA transcripts. Single-stranded RNA (ssRNA) probes (mRNA) were prepared by in vitro tran-

TABLE 1. Homotypic and heterotypic neutralizing antibody titers of reference antisera used for serotyping rotavirus isolates Anti-

Immu-

serum

nizing

no.

virus

786 293 285 298

Wa DS-1 P ST-3

a

Antiserum titer

Sero-

type 1 2 3 4

Wa

DS-1

80,OOOa

50 40,000a 50 100

600 75 300

P

300 800

75,000a 200

ST-3 300 300 300

400,000a

Titer to the homologous virus.

scription of rotavirus single-shelled particles in 250 ,ul of 70 mM Tris acetate buffer (pH 8.0) that contained 20 mM magnesium acetate, 100 mM sodium acetate, 8 mM ATP, 0.5 mM GTP, 2.5 mM CTP, 2.5 mM UTP, 0.5 mM S-adenosylmethionine, 0.1% bentonite, and [32P]GTP (25 ,uCi per reaction). After 6 h of incubation at 42°C, ssRNAs were purified by phenol-chloroform extraction and lithium chloride precipitation. RNA-RNA hybridization. The 32P-labeled ssRNA probes from Wa, KUN, and AU-1 strains were hybridized to the denatured genomic RNAs from strains 456 and 248. Likewise, the ssRNA probes prepared from strains 456 and 248 were hybridized to the denatured genomic RNAs from Wa (subgroup II, serotype 1), MO (subgroup II, serotype 3), DS-1 (subgroup I, serotype 2), KUN (subgroup I, serotype 2), and AU-1 (subgroup I, serotype 3) strains. Denaturation of dsRNA (approximately 1 ,ug) was accomplished by 2 min of incubation at 100°C followed by quenching on ice for 2 min, and to this were added 32P-labeled probes (10,000 cpm for each denatured dsRNA). Hybridization was allowed to occur at 65°C for 16 h in a buffer containing 5 mM Tris acetate, 150 mM NaCl, 1 mM EDTA, and 0.1% sodium dodecyl sulfate (pH 7.5). After hybridization, the RNAs were precipitated with ethanol and dissolved in a sample buffer containing 62.5 mM Tris hydrochloride (pH 6.8), 5% (vol/vol) 2-mercaptoethanol, 10% (vol/vol) glycerol, 2% (wt/ vol) sodium dodecyl sulfate, and 0.001% (wt/vol) bromophenol blue. The resulting hybrids, consisting of negative-strand genomic RNA and the positive-strand probe, were separated on a 10% polyacrylamide gel with a 4% stacking gel and then were stained with ethidium bromide. Autoradiographs were prepared by exposing dried gels to X-Omat AR films (Eastman Kodak Co., Rochester, N.Y.) at -80°C. RESULTS Selection of rotavirus isolates. A total of 454 episodes of rotavirus-associated diarrhea were detected in the Matlab region of Bangladesh between 14 January 1985 and 31 December 1986, and 381 rotavirus isolates were successfully culture adapted (Ward et al., Abstr. Sixth Int. Conf. Comp. Appl. Virol. 1989). Electrophoretic analysis of viral RNA segments revealed that 335 isolates composed of at least 79 distinct electropherotypes had 11 electrophoretically identical segments in both the initial stool and culture-adapted preparations indicative of infection with a single virus strain. The serotypes of these 335 isolates were determined by neutralization by using antisera to human rotavirus strains Wa, DS-1, P, and ST-3, representative of serotypes 1 through 4, respectively. Cross-neutralization analyses showed that these antisera were highly serotype specific

(Table 1). All 335 culture-adapted isolates were neutralized by at least one reference antiserum, but 9 were weakly neutralized

VOL. 64, 1990

NATURAL REASSORTANTS OF HUMAN ROTAVIRUSES

Wa

Wa

248 456

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FIG. 1. Electrophoretic analyses of genomic RNA segments of the new human rotavirus isolates 248 and 456 in relation to the prototype strains Wa and DS-1.

6

78

by one or more additional reference sera (data not shown). One possible explanation for this observation was that these nine isolates, all of which had long electrophoretic patterns, were reassortants that contained gene segments encoding the VP4 and VP7 proteins inherited from rotaviruses of different serotypes. Surprisingly, for seven of these nine isolates, one of the neutralizing antisera was to the DS-1 (serotype 2) strain. It is, therefore, likely that if these seven are intertypic reassortants, their parents were from different genogroups. One of these isolates (strain 248) was further characterized to make this determination. In addition to these seven isolates, two others also had properties that suggested they might be reassortants of strains belonging to different genogroups. One belonged to serotype 2 but had a long electrophoretic pattern, while the other was identified as a serotype 4 strain with a short electrophoretic pattern. The former (strain 456) was further characterized for genogroup determination. TABLE 2. Subgroup and serotypic analyses of rotavirus isolates 456 and 248 Isolate

456 248

Subgroup

II II

Neutralization titer with reference antiserum 786 (Wa)

293 (DS-1)

285 (P)

298 (ST-3)

18% mismatch) (28) genetic homology in this particular segment (possibly segment 7 or 8) to form stable hybrids, but both had greater homology with this segment of prototype strains belonging to the DS-1 genogroup. Although only 2 isolates out of 335 culture-adapted strains obtained during the study periods were examined by RNARNA hybridization, properties of 7 other isolates suggested that they may also have been intergenogroup reassortants. Reports by other investigators support the suggestion that natural intergenogroup reassortants may not be as uncommon as originally believed (2, 4, 25, 27, 36). The finding that almost all human rotaviruses examined in previous studies involving large numbers of isolates were readily separated into three groups based on serotype, subgroup, and electropherotype, however, still supports the prediction that intergenogroup reassortants are relatively rare, perhaps because they are less stable than reassortants with segments derived from viruses from within a single genogroup. ACKNOWLEDGMENTS

We thank investigators at the International Center for Diarrheal Disease Research, Bangladesh, for their efforts in procurement of the stool specimens from which the rotaviruses examined in this study were isolated. We also thank D. Bernstein for critical reading of the manuscript and D. Sexton for assistance in its preparation. LITERATURE CITED

1. Aboudy, Y., I. Shif, I. Zilberstein, and T. Gotlieb-Stematsky. 1988. Use of polyclonal and monoclonal antibodies and analysis of viral RNA in the detection of unusual group A human rotaviruses. J. Med. Virol. 25:351-359. 2. Ahmed, M. U., K. Taniguchi, N. Kobayashi, T. Urasawa, F. Wakasugi, M. Islam, H. Shaikh, and S. Urasawa. 1987. Characterization by enzyme-linked immunosorbent assay using subgroup-

and serotype-specific monoclonal antibodies of human

rotavirus obtained from diarrheic patients in Bangladesh. J. Clin. Microbiol. 27:1678-1681. 3. Albert, M. J., L. E. Unicomb, and R. F. Bishop. 1987. Cultivation and characterization of human rotaviruses with "super short" RNA patterns. J. Clin. Microbiol. 25:183-185. 4. Beards, G. M., U. Desselberger, and T. H. Flewett. 1989. Temporal and geographical distribution of human rotavirus serotypes, 1983 to 1988. J. Clin. Microbiol. 27:2827-2833.

4a.Bernstein, D. I., M. A. Kacica, M. M. McNeal, G. M. Schiff, and R. L. Ward. 1989. Local and systemic antibody response to rotavirus WC3 vaccine in adult volunteers. Antiviral Res. 12: 293-300. 5. Brown, D. W. G., M. M. Mathan, M. Mathew, R. Martin, G. M. Beards, and V. I. Mathan. 1988. Rotavirus epidemiology in Vellore, South India: group, subgroup, serotype, and electropherotype. J. Clin. Microbiol. 26:2410-2414. 6. Chanock, S. J., E. A. Wenske, and B. N. Fields. 1983. Human rotaviruses and genome RNA. J. Infect. Dis. 148:49-50. 7. Clark, H. F., Y. Hoshino, L. M. Bell, J. Groff, G. Hess, P.

Bachman, and P. A. Offit. 1987. Rotavirus isolate WI61 senting a presumptive new human serotype. J. Clin. Microbiol. 25:1757-1762. 8. Estes, M. K., and J. Cohen. 1989. Rotavirus gene structure and function. Microbiol. Rev. 53:410-449. 9. Flores, J., Y. Hoshino, E. Boeggeman, R. Purcell, R. M. Chanock, and A. Z. Kapikian. 1986. Genetic relatedness among repre-

animal rotaviruses. Arch. Virol. 87:273-285. 10. 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 determined by RNA hybridization. Infect. Immun. 37:648-655. 11. Flores, J., I. Perez-Schael, E. Boeggeman, L. White, M. Perez, R. Purcell, Y. Hoshino, K. Midthun, R. M. Chanock, and A. Z. Kapikian. 1985. Genetic relatedness among human rotaviruses. J. Med. Virol. 17:135-143. 12. Garbarg-Chenon, A., F. Bricout, and J. C. Nicholas. 1984. Study of genetic reassortments between two human rotaviruses. Virology 139:358-365. 13. Georges-Courbot, M. C., A. M. Beraud, G. M. Beards, A. D. Campbell, J. P. Gonzalez, A. J. Georges, and T. H. Flewett. 1988. Subgroups, serotypes, and electropherotypes of rotavirus isolated from children in Bangui, Central African Republic. J. Clin. Microbiol. 26:668-671. 14. Ghosh, S. K., and T. N. Naik. 1989. Detection of a large number of subgroup 1 human rotaviruses with a "long" RNA electropherotype. Arch. Virol. 105:119-127. 15. Greenberg, H. B., J. Flores, A. R. Kalica, R. G. Wyatt, and R. Jones. 1983. Gene coding assignments for growth restriction, neutralization and subgroup specificities of the W and DS-1 strains of human rotavirus. J. Gen. Virol. 64:313-320. 16. Greenberg, H. B., V. McAuliffe, J. Valdesuso, R. G. Wyatt, J. Flores, A. Kalica, Y. Hoshino, and N. Singh. 1983. Serological analysis of the subgroup protein of rotaviruses, using monoclonal antibodies. Infect. Immun. 39:91-99. 17. Greenberg, H. B., J. Valdesuso, K. van Wyke, K. Midthun, M. Walsh, V. McAuliffe, R. G. Wyatt, A. R. Kalica, J. Flores, and Y. Hoshino. 1983. Production and preliminary characterization of monoclonal antibodies directed at two surface proteins of rhesus rotavirus. J. Virol. 47:267-275. 18. 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 82:8701-8704. 19. Hoshino, Y., R. G. Wyatt, H. B. Greenberg, J. Flores, and A. Z. Kapikian. 1984. Serotypic similarity and diversity of rotaviruses of mammalian and avian origin as studied by plaque reduction neutralization. J. Infect. Dis. 149:694-702. 20. Kalica, A. R., H. B. Greenberg, R. T. Espejo, J. Flores, R. G. Wyatt, A. Z. Kapikian, and R. M. Chanock. 1981. Distinctive ribonucleic acid patterns of human rotavirus subgroups 1 and 2. Infect. Immun. 33:958-961. 21. Kalica, A. R., H. B. Greenberg, R. G. Wyatt, J. Flores, M. M. Sereno, A. Z. Kapikian, and R. M. Chanock. 1981. Genes of human (strain Wa) and bovine (strain UK) rotavirus that code for neutralization and subgroup antigens. Virology 112:385-390. 22. Kapikian, A. Z., W. L. Cline, H. B. Greenberg, R. G. Wyatt, A. R. Kalica, C. E. Banks, H. D. James, Jr., J. Flores, and R. M. Chanock. 1981. Antigenic characterization of human and animal rotaviruses by immune adherence hemagglutination assay (IAHA): evidence for distinctness of IAHA and neutalizaiton antigens. Infect. Immun. 33:415-425. 23. Kitaoka, S., T. Nakagomi, N. Fukuhara, Y. Hoshino, H. Suzuki, 0. Nakagomi, A. Z. Kapikian, T. Ebina, T. Konno, and N. Ishida. 1987. Serologic characteristics of a human rotavirus isolate, AU-1, which has a "long" RNA pattern and subgroup I specificity. J. Med. Virol. 23:351-357. 24. Kutsuzawa, T., T. Konno, H. Suzuki, T. Ebina, and N. Ishida. 1982. Two distinct electrophoretic migration patterns of RNA segments of human rotaviruses prevalent in Japan in relation to their serotypes. Microbiol. Immunol. 26:271-273. 25. Mascarenhas, J. D. P., A. C. Linhares, Y. B. Gabbay, R. B. de Freitas, E. Mendez, S. Lopez, and C. F. Arias. 1989. Naturally occurring serotype 2/subgroup II rotavirus reassortants in Northern Brazil. Virus Res. 14:235-240. 26. Matsuno, S., A. Hasegawa, A. Mukoyama, and S. Inouye. 1985. A candidate for a new serotype of human rotavirus. J. Virol. 54:623-624. 27. Matsuno, S., A. Mukoyama, A. Hasegawa, K. Tanaguchi, and S.

VOL. 64, 1990

28.

29.

30.

31.

32. 33.

34.

35.

NATURAL REASSORTANTS OF HUMAN ROTAVIRUSES

Inouye. 1988. Characterization of a human rotavirus strain which is possibly a naturally-occurring reassortant virus. Virus Res. 10:167-175. Nakagomi, O., T. Nakagomi, K. Akatani, and N. Ikegami. 1989. Identification of rotavirus genogroups by RNA-RNA hybridization. Mol. Cell. Probes 3:251-261. Nakagomi, O., T. Nakagomi, Y. Hoshino, J. Flores, and A. Z. Kapikian. 1987. Genetic analysis of a human rotavirus that belongs to subgroup I but has an RNA pattern typical of subgroup II human rotaviruses. J. Clin. Microbiol. 25:11591164. Nakagomi, O., T. Nakagomi, H. Oyamada, and T. Suto. 1985. Relative frequency of human rotavirus subgroups 1 and 2 in Japanese children with acute gastroenteritis. J. Med. Virol. 17:29-34. Nakagomi, O., H. Oyamada, S. Kuroki, Y. Kobayashi, A. Ohshima, and T. Nakagomi. 1989. Molecular identification of a novel human rotavirus in relation to subgroup and electropherotype of genomic RNA. J. Med. Virol. 28:163-168. Nakagomi, T., and 0. Nakagomi. 1989. RNA-RNA hybridization identifies a human rotavirus that is genetically related to feline rotavirus. J. Virol. 63:1431-1434. Offit, P. A., and G. Blavat. 1986. Identification of the two rotavirus genes determining neutralization specificities. J. Virol. 57:376-378. Rodger, S. M., R. F. Bishop, C. Birch, B. McLean, and I. H. Holmes. 1981. Molecular epidemiology of human rotaviruses in Melbourne, Australia, from 1973 to 1979, as determined by electrophoresis of genome ribonucleic acid. J. Clin. Microbiol. 13:272-278. Sethi, S. K., D. M. Olive, 0. 0. Strannagard, and W. Al-Nakib. 1988. Molecular epidemiology of human rotavirus infections based on genome segment variation in viral strains. J. Med. Virol. 26:249-259.

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36. Steele, A. D., and J. J. Alexander. 1988. The relative frequency of subgroup I and II rotaviruses in black infants in South Africa. J. Med. Virol. 24:321-327. 37. Svensson, L., I. Uhnoo, M. Grandien, and G. Wadeil. 1986. Molecular epidemiology of rotavirus infection in Uppsala, Sweden, 1981: disappearance of a predominant electropherotype. J. Med. Virol. 18:101-111. 38. Taniguchi, K., S. Urasawa, and T. Urasawa. 1985. Preparation and characterization of neutralizing monoclonal antibodies with different reactivity patterns to human rotaviruses. J. Gen. Virol. 66:1045-1053. 39. Taniguchi, K., T. Urasawa, S. Urasawa, and T. Yasuhara. 1984. Production of subgroup-specific monoclonal antibodies to an enzyme-linked immunosorbent assay for subgroup determination. J. Med. Virol. 14:115-125. 40. Urasawa, S., T. Urasawa, and K. Taniguchi. 1986. Genetic reassortment between two human rotaviruses having different serotype and subgroup specificities. J. Gen. Virol. 67:15511559. 41. Ward, R. L., and D. R. Knowlton. 1989. Genotypic selection following coinfection of cultured cells with subgroup 1 and subgroup 2 human rotaviruses. J. Gen. Virol. 70:1691-1699. 42. Ward, R. L., D. R. Knowlton, and P.-F. L. Hurst. 1988. Reassortant formation and selection following coinfection of cultured cells with subgroup 2 human rotaviruses. J. Gen. Virol. 69:149-162. 43. Ward, R. L., D. R. Knowlton, and M. J. Pierce. 1984. Efficiency of human rotavirus propagation in cell culture. J. Clin. Microbiol. 19:748-753. 44. Ward, R. L., D. R. Knowlton, G. M. Schiff, Y. Hoshino, and H. B. Greenberg. 1988. Relative concentrations of serum neutralizing antibody to VP3 and VP7 proteins in adults infected with a human rotavirus. J. Virol. 62:1543-1549.