G6 (11), and the Silenus set consisted of MAbs specific for Gl to G4 (Silenus Laboratories, Victoria, Australia). For the Sao. Paulo study, stool specimens from ...
CLINICAL MICROBIOLOGY, May 1994,
Vol. 32, No. 5
0095-1137/94/$04.00+0 Copyright X 1994, American Society for Microbiology
Rotavirus Serotype G5 Associated with Diarrhea in Brazilian Children VERA GOUVEA,l 2* LIANE DE CASTRO,1 MARIA DO CARMO TIMENETSKY,2'3 HARRY GREENBERG,4 AND NORMA SANTOS2 Department of Virology, Instituto Oswaldo Cruz, FIOCRUZ, Rio de Janeiro, 1 and Virology Laboratory, Instituto Adolfo Lutz, Sao Paulo,3 Brazil; Division of Microbiology, Food and Drug Administration, Washington, D.C. 202042; and Division of Gastroenterology, Stanford University, Stanford, Califomia 943054 Received 18 November 1993/Returned for modification 27 January 1994/Accepted 10 February 1994
Rotavirus serotype G5 in fecal specimens of 38 Brazilian children with diarrhea was identified by PCR and enzyme immunoassays. The strains exhibited long RNA electropherotypes and either subgroup II or nonsubgroup I-nonsubgroup II specificities. Serotype G5 has been found in piglets and horses but not yet in humans.
Group A rotaviruses are important and widespread agents of gastroenteritis in children and in the young of many animal species (3). Of the 14 distinct rotavirus G serotypes, 7 have been described as human pathogens: the widespread and well-studied types Gl to G4 (for which vaccines are being developed) and the less commonly found types G8, G9, and G12. However, rotavirus strains of serotypes G6 and G10, which were thought to be exclusively bovine pathogens, have recently been recovered from children with gastroenteritis (4, 13). We describe here the finding of several rotavirus G5 strains associated with diarrhea in Brazilian children over the last decade. Serotype G5 has been found predominantly in swine and occasionally in horses; however, to our knowledge it has not been previously recovered from humans. Stool specimens were collected from 1983 to 1992 during two surveys of the etiology of acute gastroenteritis in children under 5 years of age in Brazil. Specimens in the nationwide study were screened for rotavirus at 25 participating regional laboratories by enzyme immunoassay (EIA) and RNA electropherotyping (polyacrylamide gel electrophoresis [PAGE]) (8, 12). Of the rotavirus-positive specimens, 329 were further analyzed in EIAs for subgroup (7) and serotype at Fundac,ao Oswaldo Cruz (FIOCRUZ) (2). Two sets of neutralizing monoclonal antibodies (MAbs) were used for serotyping; the Stanford set consisted of MAbs specific for Gl to G3, G5, and G6 (11), and the Silenus set consisted of MAbs specific for Gl to G4 (Silenus Laboratories, Victoria, Australia). For the Sao Paulo study, stool specimens from diarrheic children were collected at hospitals, pediatric clinics, and day-care centers and were sent to Instituto Adolfo Lutz for screening by EIA and PAGE. Those positive for rotavirus (170 specimens) were genotyped at the Food and Drug Administration by an original PCR typing assay that uses the H pool of primers specific for Gl to G4 and G9 (5) and a newly developed assay that uses the A pool of primers specific for G5, G6, G8, and G10 to G 1 (6). The specimens were also analyzed for subgroup and serotype specificities with the Stanford reagents. A total of 38 isolates were identified as serotype G5; 15 among the FIOCRUZ specimens were identified by MAb EIA, and 23 were identified by PCR typing assays and were later confirmed by MAb EIA. These specimens reacted exclusively with the G5-specific MAb 5B8 in EIAs and with the A pool of primers in PCR typing assays, producing the expected 780-bp segment characteristic of G5 strains (Fig. 1). * Corresponding author. Mailing address: 1220 North Pierce Street, no. 701, Arlington, VA 22209. Phone: (703) 522-8431.
Although all rotavirus G5 isolates presented long electropherotypes, considerable variations, particularly in the G7 to G9 triplet, were seen. Two-thirds of the isolates demonstrated subgroup II specificity, whereas the remaining one-third demonstrated neither subgroup I nor subgroup II specificity (Table 1). Three specimens were identified as both subgroup I and subgroup II; however, they contained multiple rotaviruses, as demonstrated by dual G5 and G2 specificities by both PCR and EIA typing tests and by extra RNA segments demonstrated by PAGE. Because G2 strains are usually subgroup I, it may be assumed that the G5 isolates present in these mixtures bear a single subgroup II specificity, as do most of the G5 isolates found in this study. Serotype G5 showed wide temporal and geographic distributions in the Brazilian population. It has circulated in the central plains of Goias, the northeast state of Pernambuco, and the overpopulated southeast regions of Rio de Janeiro and Sao Paulo during the last decade (Table 1). Although serotype G5 was found predominantly in sporadic cases of diarrhea, it was apparently associated with two outbreaks of gastroenteritis. Three G5 isolates were recovered from severely ill children in Valenca, Rio de Janeiro, during an outbreak of diarrhea in 1983 that involved many adults. Two other G5 isolates were from an outbreak in a day-care center in Sao Paulo, in which three children with severe dehydration were hospitalized. Secondary cases that followed involved the father and older siblings of a child attending the same day-care center. Of the 329 rotavirus-positive specimens analyzed at FIOCRUZ, 15 (4.6%) were identified as serotype G5. A similar rate of G5 isolates (5.5%) was found in the survey by the Instituto Adolfo Lutz from 1983 to 1991; however, the rate was surprisingly high (26%) in 1992. Until 1991, most patients were from the Sao Paulo metropolitan area; however, by 1992, most of the specimens came from surveillance centers in other cities of the state that had joined the study that year. The increased frequency of the G5 serotype possibly reflected a shift in the type of community being studied, i.e., from a predominantly urban to a more rural setting. Rotaviruses have a wide host range and apparently no absolute host species restrictions, although experimental infections with heterologous viruses are less severe and often asymptomatic (3). These features formed the basis for a Jennerian approach to rotavirus vaccination. Recently, strong evidence for occasional interspecies transmission of rotavirus from domestic animals to humans under natural conditions has accumulated (10). Strains that are genetically closely related to feline and canine (9) or bovine (4, 13) rotaviruses have been recovered from children with both asymptomatic and symp1408
VOL. 32, 1994
results suggest that they are most likely natural reassortants between animal G5 strains and human strains rather than purely animal strains. In a seroepidemiologic study in Ecuador, Brussow et al. (1) found G5-specific neutralizing antibodies in 6% of children naturally exposed to rotavirus. Four serum samples neutralized the G5 strain OSU exclusively, thus suggesting that anti-G5 antibodies had been elicited against true infections with G5 strains rather than against repetitive infections with heterotypic viruses that would have resulted in the broadening of the children's immune responses. Our findings that serotype G5 rotaviruses infect humans naturally and may produce severe gastroenteritis in young children have obvious implications for the planning and development of effective vaccines against rotavirus disease. N.S. is the recipient of a fellowship from CAPES, Brasilia, Brazil.
FIG. 1. PCR typing assay with the H pool of primers specific for serotypes GI to G4 and G9 (lanes 1, 3, 5, and 7) or the A pool of primers specific for serotypes G5, G6, G8, G10, and GIl (lanes 2, 4, 6, and 8) of the copied vp7 gene of a G3 isolate (lanes 1 and 2), the prototype G5 porcine OSU strain (lanes 3 and 4), and two human G5 isolates (isolate A [lanes 5 and 6] and isolate B [lanes 7 and 8]). Lanes M, 100-bp marker with highlighted 600-bp segment (Bethesda Research Laboratories, Gaithersburg, Md.).
tomatic infections. Such strains have demonstrated subgroup I specificity and a long RNA pattern, which is an unusual combination for human strains but one that is characteristic of animal strains, including all known animal G5 strains. However, the G5 isolates found in our study presented a long RNA pattern and subgroup II specificity (although some isolates demonstrated neither subgroup I nor subgroup II specificity), a combination that is almost exclusively found in rotaviruses of human origin. Furthermore, preliminary analysis of other genes of the human G5 isolates has indicated that their gene 4 is closely related to those commonly found in human rotaviruses and is distinct from that of the porcine OSU strain. These
8. TABLE 1. Distribution of the 38 rotavirus G5 isolates recovered
from children with diarrhea in Brazil
No. of specimens that demonstrated the
following specificities: State
Rio de Janeiro
1983 1985 1986 1987 1989 1990 1991 1992 1991 1983 1991
2 1 0 0 1 1 0 11 2 2 3
Mixed infections with G2 strains.
Subgroup I II+ subgroup
0 0 0 0 0 2
0 2 1 0 1 0 6 0 0 1
0 0 0 0
REFERENCES Brussow, H., P. A. Ollit, and J. Sidoti. 1991. Neutralizing antibodies to heterologous animal rotavirus serotypes 5, 6, 7, and 10 in sera from Ecuadorian children. J. Clin. Microbiol. 29:869-873. de Castro, L. 1993. Subgroups, serotypes and electropherotypes of rotaviruses circulating in Brazil. M.S. thesis. FIOCRUZ, Rio de Janeiro, Brazil. Estes, M. K., E. L. Palmer, and J. F. Obijeski. 1983. Rotavirus: a review. Curr. Top. Microbiol. Immunol. 105:123-184. Gerna, G., A. Sarasani, M. Parea, S. Arista, P. Miranda, H. Brussow, Y. Hoshino, and J. Flores. 1992. Isolation and characterization of two distinct human rotavirus strains with G6 specificity. J. Clin. Microbiol. 30:9-16. Gouvea, V., R. I. Glass, P. Woods, K. Taniguchi, H. F. Clark, B. Forrester, and Z.-Y. Fang. 1990. Polymerase chain reaction amplification and typing of rotavirus nucleic acid from stool specimens. J. Clin. Microbiol. 28:276-282. Gouvea, V., M. C. Timenetsky, and N. Santos. 1994. Identification of bovine and porcine rotavirus G types by PCR. J. Clin. Microbiol. 32:1338-1340. Greenberg, H., V. McAulife, J. Valdesuso, R. Wyatt, J. Flores, A. Kalica, Y. Hoshino, and N. Singh. 1983. Serological analysis of the subgroup protein of rotavirus, using monoclonal antibodies. Infect. Immun. 39:91-99. 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. 16:473-477. Li, B., H. F. Clark, and V. Gouvea. 1994. Amino acid sequence similarity of the VP7 protein of human rotavirus HCR3 to that of canine and feline rotaviruses. J. Gen. Virol. 75:215-219. Nakagomi, O., and T. Nakagomi. 1993. Interspecies transmission of rotaviruses studied from the perspective of genogroup. Microbiol. Immunol. 37:337-348. Padilla-Noriega, L., C. F. Arias, S. Lopez, F. Puerto, D. R. Snodgrass, K. Taniguchi, and H. B. Greenberg. 1990. Diversity of rotavirus serotypes in Mexican infants with gastroenteritis. J. Clin. Microbiol. 28:1114-1119. Pereira, H. G., R. S. Azeredo, J. P. Leite, Z. P. Andrade, and L. de Castro. 1985. A combined enzyme immunoassay for rotavirus and adenovirus (EIERA). J. Virol. Methods 10:21-28. Urasawa, S., A. Hasegawa, T. Urasawa, K. Taniguchi, F. Wakasugi, H. Suzuki, S. Inouye, B. Pongprot, J. Supawadee, S. Suprasert, P. Rangsiyanond, S. Tonusin, and Y. Yamazi. 1992. Antigenic and genetic analyses of human rotaviruses in Chiang Mai, Thailand: evidence for a close relationship between human and animal rotaviruses. J. Infect. Dis. 166:227-234.