Molecular epidemiology of rotaviruses associated with pediatric diarrhea in Bangkok, Thailand.

Rotavirus diarrhea in 453 pediatric patients (29.8% of 1,518) was studied in greater Bangkok during 1985 to 1987. The disease persisted all year, increasing in incidence from August to January (30 to 50%). Polyacrylamide gel electrophoresis of rotavirus RNA from these patients and from an additional 46 patients of a 1982 to 1983 epidemic revealed 26 electropherotypes, 4 with short (S) and 22 with long (L) RNA profiles. Of the analyzed specimens, 85.5% were L forms. Only one or a few electropherotypes predominated in each epidemic, whereas others appeared sporadically at low frequencies. Shifts in the predominant electropherotypes were observed in every epidemic. Of these, 126 strains were tested for subgroup and serotype by monoclonal antibody enzyme immunoassay. Serotype 4 prevailed from 1982 to 1983, while serotype 1 was encountered more frequently than serotypes 2 and 4 from 1985 to 1987. A complete correlation was found between the electrophoretic migration of segments 10 and 11 and the serologically defined subgroup specificity. Distinct electropherotypes occurred within the same serotype, and strains with the identical electropherotype always showed the same serotype specificity. No specific electropherotype or serotype correlated with patient age. In this study, atypical rotaviruses and mixed infections with different rotaviruses were identified.     

Occurrence of changes in human rotavirus serotypes with concurrent changes in genomic RNA electropherotypes.

To investigate the serotypic and genetic diversity of human rotavirus strains, we have tested 513 and 519 fecal rotavirus specimens, respectively, by an enzyme-linked immunosorbent assay with serotype-specific monoclonal antibodies and by polyacrylamide gel electrophoresis of the segmented RNA genome. Of the 513 specimens, 375 were typed as serotype 1 (47.3%), serotype 2 (2.9%), serotype 3 (2.9%), or serotype 4 (17.7%). In addition, a presumptive new human serotype, tentatively referred to as serotype X in this paper, was found in 1.6% of the specimens tested. The remaining 138 specimens (26.9%) were untypeable. Considerable variation in relative frequency of circulating serotypes was observed with respect to geographic locations and observation periods. Rotavirus RNAs were visualized in 481 of 519 specimens tested. Of these, 415 were typed as 33 electropherotypes, many of which were infrequently detected and were restricted to single epidemics. Analysis of the 291 specimens whose electropherotypes and serotypes were available indicated clearly that a given RNA pattern always corresponded to a particular serotype. Heterogeneity of electropherotypes within a serotype was similarly observed in strains belonging to the four previously established serotypes. The results obtained in this study indicated that antigenic changes on the major neutralization antigen occurred always with concurrent changes of genomic RNA electropherotypes. On the other hand, serotypic changes could not be predicted from the changes in RNA electropherotypes.     

Molecular epidemiology of human rotavirus infections based on genome segment variations in viral strains

Molecular epidemiology of rotavirus infections in 621 hospitalized children was investigated by analysis of migration patterns of viral genomic ribonucleic acid (RNA) segments by electrophoresis in polyacrylamide gels. Based on migration patterns of RNA segments of 184 rotavirus strains, seven different electropherotypes were identified: 146 (79.3%) strains were "long," and 38 (20.7%) were "short" electropherotypes; 61% belonged to a single dominant "long" electropherotype, which persisted throughout the 15-month period of study, whereas six other cocirculating types appeared at varying intervals. Electrophoretic migration patterns of RNA from viral isolates of two patients suggested mixed infections with different rotaviruses. There was a lack of correlation between the electrophoretic migration of segments 10 and 11 and serologically defined subgroup specificity in three of the rotavirus strains. Rotavirus infections and different electropherotypes were observed throughout the year.     

Molecular epidemiology of rotavirus diarrhea among children and adults in Nepal: detection of G12 strains with P[6] or P[8] and a G11P[25] strain

In anticipation of a rotavirus vaccine in Nepal, this study was undertaken to determine the distribution of the G and P serotypes and electropherotypes of rotaviruses in order to examine if there is any emerging serotype or unusual strain circulating in children and adults in Nepal. Of 1,315 diarrheal stool specimens, rotavirus was detected by an enzyme-linked immunosorbent assay in 116 (17%) of 666 patients less than 5 years of age, in 18 (7%) of 260 patients 5 to 14 years of age, and in 19 (5%) of 358 patients 15 years of age and older. Approximately 75% of rotavirus diarrhea occurred in children less than 5 years of age. Approximately 70% of rotaviruses found in each of the three age groups belonged to serotype G1P[8]. Interestingly, there were 29 (20%) G12 rotaviruses carrying either P[8] or P[6] and one (0.7%) G11 rotavirus carrying an unusual P[25] genotype. RNA polyacrylamide gel electrophoresis discriminated 19 strains (electropherotypes), among which there were three codominant strains carrying G1P[8] and long RNA patterns. Five electropherotypes were discriminated among G12 rotaviruses, all of which had long RNA patterns. The fact that 20% of rotaviruses were G12 strains carrying either P[8] or P[6] and had multiple electropherotypes suggest that G12 strains are not more rare strains but that they pose an emerging challenge to current and future vaccines. The presence of multiple strains as defined by electropherotypes suggests the richness of the rotavirus gene pool in Nepal, where unusual strains may continue to emerge.     

Molecular identification of a novel human rotavirus in relation to subgroup and electropherotype of genomic RNA

A total of 41 stool rotavirus specimens collected from children with acute diarrhea at four different locations in Akita Prefecture, Japan, during the peak of the winter diarrhea epidemic in 1988 were analyzed by polyacrylamide gel electrophoresis of viral RNA in conjunction with subgrouping assay. We found that a single strain predominated, with cocirculating strains with less common electropherotypes at a given location, and that two different strains could predominate at geographically close but different locations even during a very limited time of the epidemic season. Furthermore, we isolated a human rotavirus strain (AU125) that was similar to the AU-1 strain in that it possessed a long RNA pattern yet belonged to subgroup I. Genetic analysis by RNA-RNA hybridization assay indicated that the AU125 strain was distinct from two previously identified human rotavirus gene groups (genogroups) represented by the Wa strain (subgroup II with long RNA electropherotype) and the DS-1 strain (subgroup I with short RNA electropherotype), but was very closely related to the AU-1 strain. These data suggest that the genetic diversity of human rotaviruses may be more extensive than was previously thought.     

Epidemiology of rotavirus strains infecting children throughout Australia during 1986–1987. A study of serotype and RNA electropherotype

Summary The epidemiology of human rotaviruses throughout Australia was studied by examining 344 rotavirus positive faecal specimens using an enzyme immunoassay incorporating serotype specific monoclonal antibodies. Specimens were collected from children less than 5 years old admitted to urban hospitals for treatment of acute diarrhoea during the winter months of 1986 and/or 1987 in Queensland, New South Wales, Victoria, Tasmania, South Australia, and Western Australia. The infecting rotavirus serotype was identified in 229 of 344 (66.6%) specimens. The predominant serotype throughout Australia was serotype 1 which was identified in 218 of 229 (95%) typable specimens. The majority (201 of 218) were identified as monotype 1a strains. Serotype 2 strains were found in Perth, Western Australia in 2 of 12 specimens collected in 1986 and in 6 of 32 specimens collected in 1987. RNA electropherotypes comprising 30 different patterns were detected after co-electrophoresis of 143 of the 218 serotyped strains. Twenty-nine electropherotypes were serologically homogeneous. One electropherotype contained strains with different monotypes including 1a, 1b, and 1d. The results show remarkable serological uniformity, associated with genetic diversity of rotavirus strains identified in widely separated areas of Australia during one winter.     

Isolation of a bovine rotavirus with a "super-short" RNA electrophoretic pattern from a calf with diarrhea

A rotavirus with a "super-short" RNA electropherotype was isolated from a calf with diarrhea and was designated VMRI strain. Segments 10 and 11 of this rotavirus migrated more slowly than did those of bovine rotavirus strains NCDV, B641, and B223. The electrophoretic pattern of the VMRI strain was similar to that reported for rotaviruses with super-short RNA electropherotypes from humans and rabbits. Northern (RNA) blot hybridization indicated that gene 11 of the VMRI strain was altered and migrated between gene segments 9 and 10. The subgroup of the VMRI strain was shown to be subgroup I. The VMRI strain of bovine rotavirus was neutralized by antisera containing polyclonal antibodies to rotavirus serotype 6 (bovine rotavirus serotype I) strains NCDV and B641 and by ascitic fluid containing monoclonal antibodies directed to VP7 of serotype 6 rotavirus. The VMRI strain was not neutralized by either polyclonal or monoclonal antibodies to strain B223 (bovine rotavirus serotype II). Collective data on the neutralization of the VMRI strain with monoclonal antibodies and polyclonal antibodies suggest that this virus is a member of the NCDV group (serotype 6) of rotaviruses (bovine rotavirus serotype I).     

Rotavirus infection of young children in two districts of Kenya from 1982 to 1983 as analyzed by electrophoresis of genomic RNA.

Employing techniques of polyacrylamide gel electrophoresis of viral RNA segments, we studied rotavirus strains and their relative contributions to rotavirus gastroenteritis epidemics in two major districts of Kenya. From early 1982 to the middle of 1983, 18 representative electropherotypes, including 6 short strains, were detected in 30 rotavirus specimens obtained from Nairobi, whereas 16, including 3 short strains, were detected in 70 virus specimens from coastal areas. With the exception of one strain, there were no identical electropherotypes between the two groups of rotaviruses obtained from these different districts. A change in predominant electropherotypes was observed in Mombasa in early 1983, and subsequently, newly occurring strains were detected in a small town along the coast when an apparent increase in gastroenteritis was observed in the district.     

Epidemiological patterns of rotaviruses causing severe gastroenteritis in young children throughout Australia from 1993 to 1996

Rotavirus strains that caused severe diarrhea in 4,634 (2,533 male) children aged less than 5 years and admitted to major hospitals in eight centers throughout Australia from 1993 to 1996 were subject to antigenic and genetic analyses. The G serotypes of rotaviruses were identified in 81.9% (3,793 of 4,634) children. They included 67.8% (from 3,143 children) serotype G1 isolates (containing 46 electropherotypes), 11.5% (from 531 children) serotype G2 isolates (27 electropherotypes), 0.8% (from 39 children) serotype G3 isolates (8 electropherotypes), and 1.6% (from 76 children) serotype G4 isolates (9 electropherotypes). G6 (two strains) and G8 (two strains) isolates were identified during the same period. G1 serotypes were predominant in all centers, with intermittent epidemics of G2 serotypes and sporadic detection of G3 and G4 strains. With the exception of two strains (typed as G1P2A[6] and G2P2A[6]) all serotype G1, G3, and G4 strains were P1A[8] and all serotype G2 strains were P1B[4]. Two contrasting epidemiological patterns were identified. In all temperate climates rotavirus incidence peaked during the colder months. The genetic complexity of strains (as judged by electropherotype) was greatest in centers with large populations. Identical electropherotypes appeared each winter in more than one center, apparently indicating the spread of some strains both from west to east and from east to west. Centers caring for children in small aboriginal communities showed unpredictable rotavirus peaks unrelated to climate, with widespread dissemination of a few rotavirus strains over distances of more than 1,000 km. Data from continued comprehensive etiological studies of genetic and antigenic variations in rotaviruses that cause severe disease in young children will serve as baseline data for the study of the effect of vaccination on the incidence of severe rotavirus disease and on the emergence of new strains.     

Cultivation and characterization of novel human group A rotaviruses with long RNA electropherotypes, subgroup II specificities, and serotype 2 VP7 genes.

During an epidemiological study of human rotavirus infections in Bangladesh, three group A strains hybridized with a serotype 2 oligonucleotide probe, but they had long RNA electropherotypes. The three strains were collected from 8- to 20-month-old infants with acute diarrhea and moderate malnutrition. By a modified isolation procedure, two strains (T-B and T-C) were adapted in MA104 cell cultures. They were identified to be subgroup II specific by enzyme-linked immunosorbent assay with subgroup I- and II-specific monoclonal antibodies and were identified by a fluorescent focus reduction neutralization assay with hyperimmune antisera to be serotype 2 specific. Further characterization of these unusual rotavirus strains needs to be carried out.     

Antigenic characterization of rotaviruses isolated in Kenya from 1982 to 1983.

The electropherotypes of human rotavirus RNAs from 100 diarrheic stool specimens collected in two major districts of Kenya from 1982 to 1983 were previously reported (Y. Chiba, C. Miyazaki, Y. Makino, L. N. Mutanda, A. Kibue, E. O. Lichenga, and P. M. Tukei, J. Clin. Microbiol. 19:579-582, 1984). Of these specimens, 25 that contained rotaviruses with different RNA electropherotypes were subjected to a virus isolation experiment with MA-104 cells, and 16 rotavirus strains were isolated. The use of an enzyme-linked immunosorbent assay with subgroup-specific monoclonal antibodies enabled us to successfully subgroup 15 isolates: 4 in subgroup I and 11 in subgroup II. By fluorescent-focus-neutralization test with serotype-specific rabbit antisera, 13 isolates could be serotyped: 7 as serotype 1, 4 as serotype 2, and 2 as serotype 3. Of the remaining three isolates, F153, F247, and G402, the former was doubly neutralizable with serotype 1 and serotype 4 antisera and the latter two were neutralizable with serotype 3 and serotype 4 antisera. Detailed analysis with the antisera against F153 and F247 and four serotype-specific, VP7-directed monoclonal antibodies suggested that F153 is a serotypic mosaic strain with serotype 4-specific VP3 and serotype 1-specific VP7 outer capsid proteins and F247 and G402 are possibly antigenic mosaic strains with serotype 3 and serotype 4 antigens. On the basis of the correspondence of the rotavirus isolate serotypes determined in this study to the electropherotypes reported previously, it was inferred that serotype 1 strains were most prevalent in two districts of Kenya from 1982 to 1983, followed by any type of serotypic mosaic strains.     

Culture adaptation and characterization of group A rotaviruses causing diarrheal illnesses in Bangladesh from 1985 to 1986.

Group A rotaviruses collected between 1985 and 1986 during comprehensive surveillance of treated diarrheal episodes occurring in a rural Bangladesh population were culture adapted and characterized by electropherotype, serotype, and subgroup. Of 454 episodes of rotavirus-associated diarrhea, rotaviruses were culture adapted from 381 (84%), and 335 contained 11 electrophoretically identical segments in unpassaged and cultured preparations. These 335 comprised 69 different electropherotypes with between 1 (32 isolates) and 79 representatives. The persistence of specific rotavirus strains within the study population, as defined by the detection of viruses with particular electropherotypes, was generally limited to a period of only a few months. All 335 isolates were serotyped by neutralization with hyperimmune antisera to prototype rotavirus strains representative of serotypes 1 to 4, i.e., Wa, DS-1, P, and ST-3. It was found that 80, 48, 119, and 88 isolates belonged to serotypes 1 to 4, respectively. The concentrations of hyperimmune antisera required to neutralize these isolates, however, were at least threefold greater than those needed to neutralize the homologous strains. Therefore, the isolates appeared to have altered neutralization epitopes from their prototype strains. Furthermore, the serotype 4 isolates were consistently shown to be much more closely related to the serotype 4B VA70 strain than the serotype 4A ST-3 strain. All but two isolates identified as serotypes 1, 3, or 4 had long electropherotypes and were subgroup II, and all but one serotype 2 isolate were subgroup I and had short electropherotypes. The three disparate strains appeared to be genetic reassortants. Evidence is presented that dual infections required for reassortant formation were not uncommon. Thus, formation of multiple reassortants may have been a cause for the observed rapid shift in viral strains within the study population.     

Development of neutralizing antibodies and group A common antibodies against natural infections with human rotavirus.

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 (greater than or equal to 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.     

Rotavirus serotype G5 associated with diarrhea in Brazilian children.

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.     

Rotavirus serotypes and electropherotypes in Finland from 1986 to 1990

Summary Four epidemic seasons of rotaviruses were studied in Helsinki during 1986–1990. This is the first Scandinavian study, where both electropherotypes and serotypes are determined. Out of 5316 fecal specimens 769 (14.5%) rotavirus positive samples were detected by electron microscopy. Of these, 645 isolates (83.9%) gave a clear RNA pattern in gel electrophoresis and they clustered into 87 electropherotypes. An illustrative number of isolates representing each electropherotype (=E-type) was serotyped using VP7 protein-specific monoclonal antibodies for serotypes G1–G4 and without exceptions, within one E-type only a single serotype specificity was found. After establishment of the serotype of each E-type, the distribution of serotypes was scored as 61.2%, 2.0%, 0.5% and 29.8% for G1–G4, respectively; 6.5% remained untypable. Two seasons had one predominant E-type (Season 1, 1986–87, and Season 3, 1988–89, 84.2% and 80.6% of rotavirus positive samples, respectively). Both were followed by a season with no predominant E-type, but several minor E-types. Altogether, 5 short E-types (13/645 samples) with serotype G2 specificity were found, most of them occurring in Season 2. Only 2 E-types (3 samples) belonged to serotype G3. Group C rotavirus was found in 8 specimens. In this study a shift in serotypes, from G1 to G4, was observed in Finland in 1988/89; a similar shift was reported in many European countries at that time.     

Occurrence of rotavirus RNA electropherotypes in the pediatric population in Slovakia

Using the method of electrophoresis in polyacrylamide gel (PAGE), the authors assessed electropherotypes of rotavirus RNA in extracts of 119 specimens of faeces of sick children and from neonates with asymptomatic rotavirus infection. In both investigated groups they detected electropherotypes of five profiles: of these three had the nature of "long" electropherotypes and were detected in 91.6% of the tested samples. In the group of sick children they recorded circulation of four electropherotypes with exchange of the dominant type of electropherotype after a three-year interval. In the group of healthy neonates they detected in the course of 21 months the same electropherotype, which was not detected in the group of sick children throughout the period of investigation. The identity of RNA profiles of strains from neonates was confirmed by co-electrophoresis.     

Group A rotavirus G type prevalence in two regions of Hungary

Summary Rotaviruses are a major cause of gastroenteritis in children worldwide. Rotaviruses are antigenically complex, with multiple serotypes (G types). The first longitudinal study of group A rotavirus serotype (G type) distribution in Hungary is reported. Neutralizing monoclonal antibodies specific for G1, G2, G3, and G4 were used in an enzyme immunoassay to determine the antigenic variation of group A rotaviruses in two collections of stool specimens assembled from 1984–1992 in Baranya County, southwest Hungary, and from 1988–1992 at the Central Hospital for Infectious Diseases in Budapest. Ninety-two percent of the 1215 virus-positive samples were typed as follows: G1 (81%), G2 (4%), G3 (1%), G4 (5%), or mixed type (1%). G1 was the predominant type during the entire study period with the exception of the 1988/1989 rotavirus season in Baranya County when G4 predominated. Among G1 strains, different electropherotypes were detected with a shift of the predominant G1 electropherotype(s) each 2 to 3 years. G typing from two longitudinal collections established regional differences within Hungary in the prevalence of rotavirus antigenic types among children with rotavirus-associated diarrhea. These are the first longitudinal rotavirus typing results for Hungary and Central Europe.     

Variation in neutralization epitopes of human rotaviruses in relation to genomic RNA polymorphism

Fecal rotaviruses collected from October 1983 to September 1984 from outpatients and inpatients attending the Royal Children's Hospital, Melbourne, Australia, with acute gastroenteritis were serotyped by enzyme immunoassay using rotavirus-neutralizing mouse monoclonal antibodies specific for serotypes 1 to 4. Application of three different serotype 1-neutralizing antibodies indicated variations in neutralization epitopes between serotype 1 rotaviruses on the major outer capsid glycoprotein, including a site shared with serotype 3 rotaviruses. The three different binding patterns observed were termed "monotypes." Comparison of monotype and serotype with genomic RNA profiles generated by gel electrophoresis of ds viral RNA indicated that each RNA electropherotype corresponded to only one monotype (1a, 1b, 1c) or serotype (3, 4). However, serotypes 1 (a and c) and 4 contained multiple electropherotypes. Greater numbers of RNA segment variations, including alteration in mobility of gene segments 7, 8, and 9, were evident between rotaviruses of different serotype or monotype than within those groups. Within limits of time and location, rotavirus neutralization epitope variations appear to correlate with RNA polymorphism. Where rotavirus epidemiology has been analyzed by year using RNA electropherotypes, only limited numbers of each RNA pattern need to be serotyped to ascertain the major serotypes in circulation.