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Rates of detection of rotavirus were compared by diagnostic assay and specimen type. For bulk stools, rates of detection by reverse transcriptase PCR (RT-PCR) and enzyme immunoassay (EIA) were similar, but 18% of healthy controls tested positive by RT-PCR. Testing of bulk stools by EIA appears to be optimum for rotavirus surveillance.

We examined sera from 42 patients 1 to 30 months of age for rotavirus immunoglobulin M (IgM), IgA, IgG, and IgG subclasses and sought to determine if serum antibody could serve as a reliable marker for prediction of disease severity. Infants in the first few months of life usually had high maternal IgG titers and, when they were infected with rotavirus, had low IgM titers or no IgM in acute-phase sera and poor seroconversions 3 weeks later, suggesting that maternal antibodies had inhibited viral replication and antibody responses. All patients > or =6 months of age had IgM in acute-phase sera, indicating that IgM is a good marker for acute rotavirus infection. IgG was the best overall predictor of an infection, as the convalescent-phase sera of 81% of the patients had a fourfold rise in the IgG titer. IgA titers in convalescent-phase sera and conversion rates were higher among patients > or =12 months of age than among children younger than 12 months. IgG1 was the predominant subclass detected in the acute-phase sera of some children and in all 28 convalescent-phase serum samples examined. Patients with preexisting acute-phase IgG titers of > or =100 or > or =200 had diarrhea that was less severe or of a shorter duration. These results indicate that serum IgG is the most reliable marker for seroconversion and is a consistent proxy for protection against severe disease.

Rotavirus causes severe morbidity in developed countries and frequent deaths (> or = 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 most common cause of severe gastroenteritis in young children, but the pathogenesis and immunity of this disease are not completely understood. To examine the host response to acute infection, we collected paired serum specimens from 30 children with rotavirus diarrhea and measured the levels of nine cytokines (interleukin-1beta [IL-1beta], IL-2, IL-4, IL-6, IL-8, IL-10, IL-12, gamma interferon [IFN-gamma], and tumor necrosis factor alpha [TNF-alpha]) using a microsphere-based Luminex Flowmetrix system. Patients with acute rotavirus infection had elevated median levels of seven cytokines in serum, and of these, the levels of three (IL-6, IL-10, and IFN-gamma) were significantly (P < 0.05) higher than those in serum from control children without diarrhea. Patients with fever had significantly (P < 0.05) higher levels of IL-6 in serum than control children, and those with fever and more episodes of diarrhea had significantly (P < 0.05) higher levels of TNF-alpha than those without fever and with fewer episodes of diarrhea. We further demonstrated a negative association (P < 0.05) between the levels of IL-2 and the number of stools on the day on which the first blood sample was collected. Finally, patients with vomiting had significantly (P < 0.05) lower levels of IFN-gamma than those without vomiting. Our pilot study provides evidence that the types and magnitudes of cytokine responses to rotavirus infection in children influence or reflect the clinical outcome of disease. These findings suggest that certain cytokines may play an important role in the pathogenesis of and the protection against rotavirus disease in children and, consequently, may provide directions and insights that could prove critical to the prevention or treatment of this important disease.

We previously observed a marked diversity of rotavirus strains and a high prevalence of the uncommon serotype G9 in a small survey of rotavirus strains collected from six centers in India. In the present study, we characterized a larger collection of strains from children hospitalized with severe diarrhea in seven Indian cities between 1996 and 1998. A total of 287 strains were G and P genotyped by reverse transcription-PCR, and some were further characterized by electropherotyping and subgrouping. Of the four strains common globally, three were found in only 43% of samples (P[8], G1, 15%; P[4], G2, 22%; P[8], G4, 6%), whereas G9 strains made up 17% of the total. Three different G9 strains were present: a P[8], G9 strain, which displayed the long electropherotype and subgroup II VP6 specificity, and two P[6], G9 strains, one with the long electropherotype and subgroup II specificity and the other with the short electropherotype and subgroup I specificity. Marked diversity was observed among strains collected from different cities and collected over time. Of the 253 strains that were fully typed, 54 (21%) had a mixed G or P genotype. Serotype G2 strains were detected more often in infections caused by single strains than in mixed infections (P < 0.05), whereas serotype G1 strains were found more often in mixed infections than in infections caused by single strains (P < 0.05). The diversity of rotavirus strains and the high prevalence of mixed infections confirm trends reported earlier and help to better characterize the strains of rotavirus circulating in India. Vaccines under development should clearly target G9 strains, and G9 should be included as one of the common global serotypes.

Rotavirus strains from 964 fecal specimens collected from children at 11 U.S. hospital laboratories from November 1997 to March 1998 and from samples collected at 12 laboratories from November 1998 to March 1999 were typed for G and P proteins. Serotype G1 was the predominant serotype in 1997-1998 (88%), followed by G2 (6.2%), G9 (3.3%), and G3 (1.5%). This pattern was similar to that seen in 1998-1999: G1 (79%), G2 (15%), G9 (3.0%), G4 (1.6%), and G3 (0.3%). Novel P[9] strains were identified in both seasons, and analysis of a 364-nucleotide fragment from gene segment 4 of one of the strains demonstrated 97.3% nucleotide identity with the prototype P3[9],G3 strain, AU1, isolated in Japan. This is the first report of a human AU1-like strain in the United States. These results reinforce our initial findings that serotype G9 persists in the United States but has not become a predominant strain and that the common serotypes G1 to G4 account for almost 90% of strains in circulation. Other uncommon strains exist in the United States but may have been overlooked before because of their low prevalence and the use of inadequate diagnostic tools.

The P (VP4) and G (VP7) genotypes of 167 group A rotavirus strains obtained during the period 1996 to 1998 from 149 children living in a suburban community in Guinea-Bissau, western Africa, were determined by the reverse transcription-PCR technique. A total of nine combinations including five different P types and five different G types were identified. The globally common genotype pairs P[8], G1; P[4], G2; P[8], G3 and P[8], G4 were underrepresented in this study area. We found a substantial year-to-year variation in the occurrence of the genotype combinations. In 1996 and 1997, P[6], G2 was the most frequent, whereas P[8], G1 was more common in 1998. The unusual type P[9], G3 and a few mixed infections were detected. Sixteen percent of the rotavirus-positive samples were nontypeable.

While only four globally important rotavirus G serotypes (1 to 4) have been documented, many studies suggest that serotype G9 viruses may be widely distributed and more important than previously recognized. We have evaluated 10 serotype G9 rotavirus-neutralizing monoclonal antibodies (MAbs) directed to VP7, which bound by direct enzyme immunoassay (EIA) to P1A[8], G9 rotaviruses F45, WI61, and AU32, for their ability to recognize the New Delhi G9 rotavirus 116E. Only one MAb (MAb F45:1) bound to P[11], G9 virus 116E to a high titer by EIA. This MAb was incorporated into an indirect EIA for G serotyping, which was validated with prototype cultivable human rotaviruses of G types 1 to 4 and 9. The EIA was compared with genotyping by reverse transcriptase PCR (RT-PCR) under code for the determination of the G types of rotaviruses obtained from neonates in New Delhi, India. The sensitivities of RT-PCR and EIA (after two additional freeze-thaw cycles) for the typing of G9 rotaviruses were 91 and 86%, respectively, for 24 culture-adapted rotavirus strains. The untypeable culture-adapted rotavirus samples also were unreactive with VP7 group antigen-reactive MAb 60. After two additional freeze-thaw cycles, only 26 of 42 (62%) of stools containing rotavirus typed as G9 by RT-PCR were positive for G9 rotavirus by EIA. Stools containing rotavirus untypeable by EIA contained significantly less MAb 60-reactive VP7 antigen (P = 0. 0001) than the stools containing typeable rotavirus. Thus, RT-PCR genotyping was the more sensitive method for determination of G9 type, but a serotype was readily determined in rotavirus samples containing MAb 60-reactive VP7 antigen by an EIA that incorporates MAb F45:1.

We characterized 1,534 rotavirus (RV) strains collected in Bangladesh from 1992 to 1997 to assess temporal changes in G type and to study the most common G and P types using reverse transcription-PCR, oligonucleotide probe hybridization, and monoclonal antibody-based enzyme immunoassay. Results from this study combined with our previous findings from 1987 to 1991 (F. Bingnan et al., J. Clin. Microbiol. 29:862-868, 1991, and L. E. Unicomb et al., Arch. Virol. 132:201-208, 1993) (n = 2,515 fecal specimens) demonstrated that the distribution of the four major G types varied from year to year, types G1 to G4 constituted 51% of all strains tested (n = 1,364), and type G4 was the most prevalent type (22%), followed by type G2 (17%). Of 351 strains tested for both G and P types, three globally common types, type P[8], G1, type P[4], G2, and type P[8], G4, comprised 45% (n = 159) of the strains, although eight other strains were circulating during the study period. Mixed G and/or P types were found in 23% (n = 79) of the samples tested. Type G9 RVs that were genotype P[6] and P[8] with both long and short electrophoretic patterns emerged in 1995. The finding of five different genotypes among G9 strains, of which three were frequently detected, suggests that they may have an unusual propensity for reassortment that exceeds that found among the common G types. We also detected antigenic changes in serotypes G2 and G4 over time, as indicated by the loss of reactivity with standard typing monoclonal antibodies. Our data suggest that a vaccine must provide protection against type G9 RVs as well as against the four major G types because G9 strains constituted 16% (n = 56) of the typeable RV strains and have predominated since 1996.

We recently established a rotavirus strain surveillance system in the United States to monitor the prevalent G serotypes before and after the anticipated implementation of a vaccination program against rotavirus and to identify the emergence of uncommon strains. In this study, we examined 348 rotavirus strains obtained in 1996 to 1997 from children with diarrhea in 10 U.S. cities. Strains were characterized for P and G types, subgroups, and electropherotypes by using a combination of monoclonal antibody immunoassay, reverse transcription-PCR, and hybridization. The four strains most commonly found worldwide comprised 83% of the isolates (P[8]G1, 66.4%; P[4]G2, 8.3%; P[8]G3, 6.9%; P[8]G4, 1.4%), but 9.2% were unusual strains (P[6]G9, 5.5%; P[8]G9, 1.7%; P[6]G1, 1.4%; and P[4]G1 and P[8]G2, 0. 3% each). Strains not typeable for P or G type accounted for 5.5% of the total, while 2.3% of the strains had more than one G type (mixed infections). All P[6]G9 strains tested had short electropherotypes and subgroup I specificity and were detected in 4 of 10 cities, while P[8]G9 strains had long electropherotypes and subgroup II VP6 antigens. Both sequence analysis of the VP7 open reading frame (about 94 to 95% amino acid identity with the VP7 gene of G9 prototype strain WI61) and binding to a G9-specific monoclonal antibody strongly suggest that U.S. G9 strains belong to serotype G9. The high detection rates of unusual rotaviruses with G9 (7.2%) or P[6] (6.9%) specificity in multiple U.S. cities suggest the emergence of new strains or inadequate diagnosis in the past. The epidemiologic importance of these strains remains to be determined.

Rotavirus (RV) strains infecting newborns often have unique neutralization antigens (P serotypes) on their outer capsids that are distinct from those found on RV strains that cause diarrhea in older children. We examined the hypothesis that unusual RV strains preferentially infect newborns because the newborns lack maternal neutralizing antibodies to these strains. To test this hypothesis, sera and saliva samples collected from neonates infected with 116E-like (P[11]G9) strains in the maternity ward of the All India Institute of Medical Sciences (AIIMS) hospital in New Delhi were tested for neutralizing antibodies against common RV strains and those infecting newborns and these titers were compared with those of newborns who did not become infected (controls). The infected neonates had significantly lower levels of cord blood neutralizing antibodies to 116E than the controls, suggesting that immunity to neonatal RV infection is acquired transplacentally through maternal antibodies. Further, this study confirmed the immunogenicity of the AIIMS neonatal strain 116E, a vaccine candidate, in its ability to evoke a potent RV-specific immunoglobulin A and neutralizing antibody response in serum and saliva among the infected babies. Our findings have important implications for the development of an effective RV vaccine. In India, where G9 strains are common in the community, the use of 116E as a vaccine, together with the rhesus tetravalent vaccine, may provide a broader protection against all the circulating RV serotypes, including serotype G9, which is not represented in the current rhesus RV tetravalent vaccine (G1-G4).

Complementation and recombination have been observed with temperature-sensitive mutants of Bunyavirus snowshoe hare virus.