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In the present investigation we molecularly characterized nontypeable rotavirus strains previously identified during surveillance in New Delhi, India. The majority of strains were demonstrated to belong to genotype G1 (54.5%) or P[8] (77.8%) on the basis of nucleotide sequencing of fragments from their VP7 and VP4 genes. The other genotypes detected included G2, G8, G9, G12, and P[4]. A G8P[6] strain, strain DS108, was detected for the first time in northern India. The VP7 gene of DS108 was most homologous with the VP7 gene of a bovine G8 strain, strain A5 (98.9%), indicating its bovine parentage. In contrast, the VP4 gene had a high degree of nucleotide sequence homology (92.9% to 99.1%) with the VP4 genes of human P[6] strains. The VP6 gene and nonstructural genes (NSP1 to NSP3 and NSP5) were most homologous with the VP6 gene and nonstructural genes of human rotaviruses belonging to the DS1 genogroup. Interestingly, the NSP4 gene of DS108 clustered within genotype E6 that until now had only two representative strains, both with G12P[6] specificity (strains RV176-00 and N26-02). Together, these results indicate that G8 strain DS108 belongs to the DS1 genogroup and could be the result of the acquisition of the VP7, VP4, and NSP4 genes by a human G2P[4] strain from more than one donor, similar to the evolution of G12P[6] strain RV176-00. The present study highlights the importance of characterizing multiple genes of nontypeable rotavirus strains to detect novel strains and get a more complete picture of rotavirus evolution.

The prospect that rotavirus diarrhea in children may soon be prevented by vaccines has placed a new priority on understanding the diversity of rotavirus strains and the mechanism by which these strains evolve over time. We have characterized a total of 465 rotavirus strains collected in North India from 2000 to 2007 for G and P types by reverse transcription-PCR and sequencing. The novel G12 rotavirus strains recently detected in other countries were first detected in India in 2001 and have emerged as the predominant strains in Delhi, India, during 2005 to 2007. While the VP7 sequence was highly homologous among G12 strains isolated in Delhi, suggesting recent emergence from a common ancestor, the strains had a diverse constellation of other gene segments, demonstrating substantial reassortment. For the entire period, the common rotavirus G types G1 (26%), G2 (25%), and G9 (14%) comprised 65% of the strains, and common P types, P[4] (19%), P[6] (22%), and P[8] (35%), comprised 76% of the total P types. Of note, we detected a high percentage of unusual (17%) strains and fecal specimens with mixed (12% G and 15% P) rotavirus infections having a variety of genomic constellations. For the first time, we identified two novel rotavirus strains with unusual G/P combinations, G2P[11] and G3P[11], in patients with diarrhea. The study highlights the great diversity among rotaviruses isolated from Indian children, the opportunity for genetic reassortment between strains, and the emergence of a novel G12 strain in our country. Due to the demonstrated effect of antigenic diversity on rotavirus vaccines, it will be important to continue careful monitoring of these strains as rotavirus vaccine programs are implemented in India.

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.

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.

A gene probe derived from the colonization factor antigen I (CFA/I) operon cross-hybridized at very low stringency to plasmid DNA from coli surface antigen 17 (CS17)-producing enterotoxigenic Escherichia coli (ETEC) and from the ETEC strain F595C, which was negative for previously described CFAs, CSs, and putative colonization factors (PCFs). A 16-kDa protein was identified in sodium dodecyl sulfate-polyacrylamide gel electrophoresis of heat extracts prepared after growth of strain F595C at 37 degrees C on CFA agar containing bile salts. Transmission electron microscopy revealed bile salt- and temperature-dependent expression of fimbriae with a diameter of 7 nm. After transformation with a recombinant plasmid harboring the cfaR gene, which encodes a positive regulator of several CFAs, PCFs, and CSs, the 16-kDa protein was hyperexpressed. Polyclonal antibodies raised against this protein bound to the fimbriae and inhibited the adhesion of F595C bacteria to tissue-cultured Caco-2 cells. Nucleotide sequence determination of the gene encoding the 16-kDa fimbrial subunit revealed a high degree of amino acid sequence homology to the CFA/I, CS1, CS2, CS4, CS14, and CS17 polypeptides. The term CS19 is proposed for the new fimbria.

An enterotoxigenic Escherichia coli (ETEC) strain producing a previously undescribed putative colonization factor was isolated from a child with diarrhea in India. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of bacterial heat extracts revealed a polypeptide band of 20.8 kDa when the bacteria were grown at 37 degrees C which was absent after growth at 22 degrees C. A specific rabbit antiserum raised against the purified 20.8-kDa protein bound specifically to the fimbriae, as shown by immunoelectron microscopy, and inhibited bacterial adhesion to tissue-cultured Caco-2 cells. Transformation with a recombinant plasmid harboring the cfaD gene, which encodes a positive regulator for several ETEC fimbriae, induced hyperexpression of the 20.8-kDa fimbrial subunit and a substantial increase in the proportion of bacterial cells that were fimbriated. The N-terminal amino acid sequence of the polypeptide showed 65 and 60% identity to the PCFO20 and 987P fimbriae of human and porcine ETEC, respectively. We propose the term CS20 for this new putative colonization factor of human ETEC.

Between April and December 1993, we determined P and G genotypes of group A rotavirus strains obtained from children admitted to diarrhea treatment centers in five Indian cities. From a total of 63 rotavirus-positive specimens, we identified 10 different strains with five different G genotypes and four distinct P types by using reverse transcription-PCR. The common worldwide strains G1P8, G2P4, G3P8, and G4P8 were underrepresented among Indian children (33%), whereas strains of P type 6 (G1P6, G2P6, G3P6, G4P6, and G9P6), which primarily infect asymptomatic newborns but are rare in children with diarrhea were common in India (43%). Of these, G9P6, a strain not previously reported to be found in children with diarrhea, was the most prevalent (22%). Eleven percent of the strains were nontypeable, and another 11% of the specimens had mixed infections. Using digoxigenin-labeled, genotype-specific hybridization probes, we confirmed all G9 strains and mixed infections tested and identified three nontypeable strains (one G9 and two P8). The epidemiological significance of G9 rotavirus strains, if confirmed in other settings, may have important implications for vaccine development.

Enteropathogenic Escherichia coli (EPEC) strains designated on the basis of their serotypes are epidemiologically associated with diarrhea. They adhere to the intestinal mucosa, producing the characteristic attaching and effacing (AE) lesion in an in vitro organ culture system. EPEC manifest localized adherence (LA) in the HEp-2 cell assay, and this is commonly used for clinical diagnosis. Recently, the fluorescence actin staining (FAS) test was proposed for the identification of E. coli causing the AE lesion. We therefore compared the FAS test with the HEp-2 cell assay and the EPEC adherence factor (EAF) probe assay for the detection of EPEC strains. Among 240 stool samples from children with diarrhea examined, 176 yielded E. coli and 14 of these strains showed the LA pattern in the HEp-2 cell assay; 11 of these were positive by both the EAF and the FAS tests. By using the HEp-2 cell assay as the "gold standard," the FAS test gave a sensitivity of 78.5% and a specificity of 100%. The three localized adherent FAS-negative strains tested subsequently were positive by the enteroaggregative E. coli DNA Probe and failed to produce the AE lesions characteristic of EPEC. When these strains were not considered, the sensitivity of the FAS test for detecting isolates that manifest LA was 100%. Against the EAF probe, the sensitivity and specificity of the FAS test were 91.6 and 100%, respectively. The FAS test avoids infrequent but nevertheless important phenotypic misclassifications in the HEp-2 cell assay, and it may therefore serve as a confirmatory test for EPEC.

Plasmid DNA from two strains of enterotoxigenic Escherichia coli harboring genes encoding coli surface antigen 4 (CS4) and from seven Indian enterotoxigenic E. coli isolates cross-hybridized at low stringency but not at high stringency with two polynucleotide probes derived from the colonization factor antigen I (CFA/I) operon. Low-stringency Southern blot hybridization of PstI-digested plasmid DNA from the seven Indian isolates yielded characteristic restriction fragment patterns, distinct from those of CS4- and CFA/I-associated plasmid DNA. Two of the Indian strains were transformed with a recombinant plasmid harboring the cfaD gene, which encodes a positive regulator of CFA/I and CS4 genes. The cfaD transformants produced large amounts of putative colonization factor O166 (PCFO166) irrespective of whether the nutrient agar contained bile salts, a growth factor otherwise required for adequate PCFO166 expression. A considerable interstrain variation in the level of PCFO166 production could be explained by differences in the proportion of bacteria that were fimbriated, as visualized by electron microscopy. The N-terminal amino acid sequence of PCFO166 fimbrial protein showed a high degree of homology with the corresponding sequences of CFA/I and CS4.

We developed an accurate nonradioactive colony hybridization assay (NCHA) using a digoxigenin-labeled polynucleotide probe and an antidigoxigenin alkaline phosphatase conjugate for the identification of enterotoxigenic Escherichia coli (ETEC) harboring genes for colonization factor antigen I (CFA/I), coli surface antigen 4 (CS4), or putative colonization factor O166 (PCFO166). In this 2-day assay, visual registration of color intensity could be used to distinguish between CFA/I-positive strains and strains with the genetic potential to express CS4 or PCFO166. A rapid NCHA was developed by which the results could be read visually 7 h and 45 min after inoculation of the bacteria. In the rapid NCHA, densitometry verified the visual discrimination between four groups of E. coli; ETEC with the CFA/I gene, ETEC with the CS4 gene, ETEC with the PCFO166 gene, and E. coli strains that lack such genes. As a confirmatory test, plasmids from ETEC with the CFA/I, CS4, or PCFO166 gene were differentiated by their characteristic restriction fragment patterns in nonradioactive Southern blot hybridization.