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Rotavirus causes severe morbidity in developed countries and frequent deaths (≥500,000 per year) in less-developed countries. Historically, four serotypes—G1, G2, G3, and G4—have predominated; they are distinguished by one of two surface neutralization antigens (VP7). However, in 1983 and 1984 we described a new rotavirus serotype, designated G9, in five children hospitalized for diarrhea in Philadelphia, Pa. G9 rotavirus was not identified again in the Western Hemisphere until it caused ca. 50% of the rotavirus disease detected in Philadelphia in the 1995-1996 season. This outbreak allowed us to question whether a rotavirus strain completely new to a well-studied community would target either very young infants or older children, cause especially severe disease, or completely displace previously extant serotypes. We observed a significant excess of G9 infections in younger infants (especially in those <6 months old) that might be attributed to the lack of G9-specific antibodies in mothers. Of further note, six of the seven oldest patients with rotavirus diarrhea were infected with the G9 strains (not significant). However, the age distribution of children with rotavirus did not differ over a 5-year study period regardless of the infecting serotype. Patients with diarrhea associated with G9 strains did not have disease more severe than that caused by the G1, G2, or G3 serotype. G9 strains did not displace the other serotypes but were virtually completely replaced by G1 or G2 serotypes in the three subsequent rotavirus seasons. We conclude that the abrupt appearance of this novel rotavirus serotype did not present a special threat to public health in the community.
Rotavirus is the leading cause of severe diarrhea among children worldwide and a priority target for vaccines development (7). The scientific basis for these vaccines has been the observation that immunity to rotavirus develops after natural infection (1, 22), and this immunity is at least partially dependent upon the serotype of the infecting strain (21). Consequently, to be effective, vaccines must protect against the main serotypes of rotavirus, and surveillance activities have been mounted by a number of groups to monitor strains and characterize the serotypes that are in circulation. In particular, vaccines prepared from animal strains of rotavirus of bovine or simian origin have been most effective when they contain reassorted gene products that encode for each individual human serotype (4, 19).
Rotaviruses are small (100-nm) icosahedral viruses with three shells that encode 11 segments of double-stranded RNA. Serotypes are determined by two gene segments that encode two proteins that form the outer capsid of the virus: VP7, the glycoprotein or G protein, and VP4, the protease-cleaved or P protein. Historically, four common G serotypes designated G1, G2, G3, and G4 have been identified worldwide. Because of the segmented nature of the genome, rotaviruses can also be characterized by the pattern of migration of their segmented genome in polyacrylamide gel electrophoresis (PAGE). For surveillance, the serotypes of rotavirus are most often determined by reverse transcription-PCR (RT-PCR) methods that provide a genetic profile that correlates well with serotype (9, 10).
As part of our effort to develop new rotavirus vaccines based upon a bovine rotavirus strain, our group has been monitoring strains of rotavirus circulating in Philadelphia from patients with diarrhea who submitted fecal specimens to the virology laboratory at the Children's Hospital of Philadelphia. Surveillance was sporadic in the 1980s but became routine in 1992 and, each season since then, a sample of all rotavirus strains has been characterized for serotype and electropherotype. Each year, we have observed the circulation of the four historically common strains, most frequently G1 and G3. Then, in the 1995-1996 season, we noted the appearance of a new strain distinguished by its uncommon short electropherotype that we designated P G9. The G9 strain, W161 (P G9), had been first observed and characterized by our group in Philadelphia in 1983 but did not reappear after 1984 (3). Since the mid-1980s, other G9 strains have been identified in Asia (strains AU32  and F45 ), and in the 1990s they have been found with increased frequency worldwide (6, 14, 20).
The emergence of an entirely new serotype of rotavirus in Philadelphia in a population without previous exposure to this novel P G9 strain provided a unique opportunity to question whether this major strain shift might, like influenza, cause an epidemic in a population without serotype-specific neutralizing antibody or, alternatively, whether preexisting immunity might protect against diarrhea caused by this heterotypic strain. Specifically, we questioned whether this novel strain might cause more severe disease in infected children or affect older children without solid immunity to this novel strain. We describe here the emergence of this novel G9 strain in Philadelphia and our studies comparing the age and clinical severity of disease in children infected with the G9 rotavirus to those of children infected with common strains.
From 1994 to 1999, fecal specimens submitted to the virology laboratory at the Children's Hospital of Philadelphia in the winter season (December to May) were screened for rotavirus by using a commercial enzyme-linked immunosorbent assay (Rotaclone; Meridian Diagnostics, Inc.). Most specimens (>80%) were obtained from patients admitted to the hospital with diarrhea, although an average of 13% were from patients whose diarrhea was nosocomial and developed >72 h after admission.
Specimens that tested positive for rotavirus were further characterized by polyacrylamide gel electrophoresis (PAGE) and RT-PCR. The strains were tested by PAGE, followed by silver staining by methods previously described (8), and strains whose 11 segments had the same migration profile were grouped for selective typing. A sample of strains from each PAGE group was then tested for G and P type by RT-PCR at the Centers for Disease Control and Prevention as reported previously (9, 12).
To assess whether patients with G9 versus patients with non-G9 strains differed in disease severity or by age at presentation, we examined all available patient records from the 1995-1996 season to collect standard information on the patient's duration and severity of diarrhea, vomiting, fever, and malaise, factors which were then scored on a scale of severity from 2 to 24 as described previously (2). These observations were also compared to those of the first 24 rotavirus disease cases identified in the previous and immediately subsequent rotavirus seasons. Scoring was performed twice, once by a physician and once by a microbiologist, and the values were then compared and averaged to provide a summary result for comparison.
The distribution of rotavirus serotypes was plotted for a sample of the specimens that were successfully electropherotyped for the five winter seasons from 1994 to 1999 (Fig. (Fig.1A).1A). Each year, the distribution of serotypes varied, with G1 strains being the most common overall (38%). In the 1995-1996 season, the new serotype P G9 strains with a short electropherotype appeared and made up a majority of the cases. The novel strain emerged without warning in January 1996, remained throughout the entire 1996 winter season, and did not reappear at the beginning of the next winter season (Fig. (Fig.1B).1B). The Philadelphia strain was indistinguishable from the subsequently isolated strain US 1205 by genogroup analysis as determined by Kirkwood et al. (13) and was extremely close to strain US 1205 by phylogenetic analysis of the VP7 gene as determined by Ramachandran et al. (18). The seasonality of rotavirus disease in the 1995-1996 season did not differ from the seasonality of rotavirus disease in the non-G9 seasons. A single additional P G9 isolate was observed in the 1997-1998 season (Fig. (Fig.1A).1A). The P genotype and electropherotype of this isolate were identical to those of the G9 rotavirus detected in the 1995-1996 season.
We hypothesized that since the community had no previous exposure or immunity to the G9 strains, children infected with the novel G9 strain might be younger or older or have more severe disease than children infected with the historically more common strains. Younger infants might be more susceptible because maternal immunity would not include G9 specific neutralizing antibodies, and older children might be overrepresented because previous infections might not have induced active immunity to the novel strain. We therefore compared the cumulative frequency distributions of ages (Fig. (Fig.2)2) and the severity scores of patients with G9 infections versus patients with non-G9 infections (Fig. (Fig.3).3). We found an excess of G9 cases among children <12 months old that was concentrated in infants <6 months of age and was statistically significant (P = < 0.01 [Kolmogorov-Smirnov goodness-of-fit test]) (15). At the other extreme of age, six of the seven oldest cases of rotavirus disease were in children infected with G9 strains, but this difference was not significant. Only 39% of all rotavirus cases fell within the age range from 6 to 23 months, which is usually considered to be the age of peak susceptibility. The age distribution of cases was compared for all children during the five seasons (Fig. (Fig.4).4). Children in the 1995-1996 season, when G9 strains were predominant, tended to be younger, with more children present who were <7 months of age, but when all infections (i.e., G9 and non-G9) were compared to other seasons, the difference was not statistically significant.
From our chart review (Fig. (Fig.3),3), we were able to develop severity scores from the records of 148 children hospitalized in the 1995-1996 season representing four different serotypes. The severity scores of children infected with G1, G3, and G9 strains did not differ (mean score, 10.0 to 11.0), but children infected with G2 strains had disease that was slightly more severe (mean score, 13.0) (15). The cumulative frequency curve of severity scores indicated that serotype G2 caused significantly more severe disease than that caused by the serotype G9 virus. The severity of disease among 24 children infected at the beginning of the preceding season and at the beginning of the post-1995-1996 season did not differ significantly from those infected in the entire 1995-1996 season.
This study documents the emergence of the novel G9 serotype of rotavirus in the US in the 1995-1996 winter season. A new and distinct P G9 strain appeared briefly in Philadelphia in 1983 (9), and different G9 strains were subsequently found in India and throughout Asia in the late 1980s and early 1990s (12). These strains have now become endemic in the United States and have established themselves as the fifth most common serotype worldwide (13, 17, 18). This emergence of a new serotype has led to the need to include new diagnostic primers for G9 strains in the RT-PCR assays to characterize rotaviruses worldwide.
The present study provides some comfort to our considering the potential efficacy of vaccines against rotavirus currently in clinical trials. We hypothesized that, like influenza, the emergence of a totally novel rotavirus strain, with both outer capsid proteins different from previously circulating strains, might lead to disease of greater severity or to breakthrough infections among older children already immune to the other common serotypes. Although we did identify an increased risk of infection in younger infants <6 months of age, perhaps due to an absence of maternal neutralizing antibody specific to this novel strain, children infected with G9 strains did not have disease of greater severity or duration than children infected with the non-G9 strains. Since initial rotavirus infections with every serotype apparently occur only when maternal antibody has waned beyond protective capacity, this observation is not surprising but is reassuring. Nonetheless, six of the seven rotavirus infections among children more than 5 years of age were caused by G9 infections. Our results are in contrast to those reported by Cubitt et al. in the United Kingdom, who found that children infected with G9 strains tended to be older and have more severe dehydration than children with non-G9 strains (5). We cannot explain this discrepancy, but we did note that the distribution of ages and clinical severity scores of our patients did not differ significantly over five winter seasons of observation in which four different G serotypes predominated.
Our results should provide some reassurance about the heterotypic protection conferred by one rotavirus infection against a second infection. Despite the complete antigenic difference in both the G and P outer capsid proteins of this novel P G9 strain, children infected with this strain, although predisposed to infection at an earlier age, did not exhibit enhanced severity of their disease. Vaccines currently under development should examine this cross-protection further.
This study was supported by a grant from Merck & Co., Inc., West Point, Pa.
We thank Philip Bennett of Merck Research Laboratories for assistance with the statistical analysis.