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J Clin Microbiol. Jul 2003; 41(7): 3100–3111.
PMCID: PMC165321

Characterization of Serotype G9 Rotavirus Strains Isolated in the United States and India from 1993 to 2001

Abstract

The emergence of rotavirus serotype G9 as a possible fifth globally common serotype in the last decade, together with its increasing detection in association with various genome constellations, raises questions about the origins and epidemiological importance of recent G9 isolates. We examined a collection of 40 G9 strains isolated in the United States from 1996 to 2001 and in India since 1993 to determine their VP7 gene sequences, P types, E types, subgroup specificities, and RNA-RNA hybridization profiles. With the exception of two U.S. strains, all of the study strains shared high VP7 gene sequence homology (<2.5% sequence divergence on both the nucleotide and amino acid levels) and were more closely related to other recent isolates than to the first G9 strains isolated in the 1980s. The VP7 gene sequence and RNA-RNA hybridization profiles of the long-E-type strains showed greater variation than the short-E-type strains, suggesting that the latter strains are the result of a relatively recent reassortment event of the G9 VP7 gene into a short-E-type lineage. No evidence for reassortment of genes other than VP4 and VP7 between major human rotavirus genogroups was observed. Except for Om46 and Om67, which formed a distinct clade, phylogenetic analysis showed that most of the study strains grouped together, with some subgroups forming according to genetic constellation, geographic location, and date of isolation. The high potential of G9 strains to generate different P and G serotype combinations through reassortment suggests that it will be important to determine if current vaccines provide heterotypic protection against these strains and underscores the need for continued surveillance for G9 and other unusual or emerging rotavirus strains.

Rotavirus is the most common etiological agent of severe diarrhea worldwide, causing an estimated 418,000 to 520,000 deaths each year, 76 to 85% of which occur in low-income countries (44). In 1998, the tetravalent rhesus rotavirus (RRV-TV) vaccine was licensed for use in the United States but was withdrawn from the market 9 months later because of its association with a rare form of bowel obstruction, intussusception (45). Although renewed efforts to produce a vaccine are under way, progress toward developing a safe, effective, and affordable vaccine has been further challenged by the extensive strain diversity of rotaviruses and the emergence of new or previously uncommon strains worldwide. These challenges underscore the need for further research on the importance of serotype-specific immunity in conferring protection against all circulating strains and continued global surveillance for rotavirus strain distribution.

The outer capsid of rotavirus is composed of two structural proteins, VP4 and VP7, which independently elicit protective neutralizing antibodies and serve as the basis of a dual serotyping system for rotavirus (30, 54). VP7, a glycoprotein, is encoded by gene 7/8/9 and specifies the G serotype. VP4, a protease-cleaved protein, is encoded by gene 4 and specifies the P serotype (18). Fourteen different G serotypes and 16 distinct P serotypes (including three subtypes) have thus far been identified, and of these, 10 G types and 10 P types have been found to occur in humans (36). In addition to the outer capsid proteins, the middle-layer major capsid protein, VP6, is also used for rotavirus classification. It determines reactivity for the seven major groups (A through G) of rotavirus and contains the antigen used to classify rotavirus into subgroups I and II. Most human infections are caused by group A rotaviruses having either subgroup I or II antigen specificity, although some have both specificities and others have neither (36). Rotavirus strains are further classified into electropherotypes (E types) on the basis of differences in the relative migration rates of genome segments in polyacrylamide gel electrophoresis (PAGE), thereby creating more opportunities for strain diversification (36). The most common major E-type patterns are designated “long” and “short” based on the fact that short-E-type strains have a striking reduction in the migration rate of segment 11 due to duplication within the 3′ untranslated region of this segment (41).

Rotavirus strain nomenclature is based on the viruses' unique combination of G type and P type; therefore, on the basis of the outer capsid proteins alone, there are potentially 100 antigenically distinct rotavirus strains capable of infecting humans. Until recently, it was thought that four common strains, including P[8]G1, P[8]G3, P[8]G4, and P[4]G2, predominated globally and that only these four strains were important targets for vaccine development (21). However, in the last decade, the intensification of rotavirus surveillance in anticipation of a vaccination program, together with the development of better characterization methods, has resulted in the detection of new (e.g., serotype G5 in Brazil) or emerging (e.g., serotype G9) human rotavirus strains and has also facilitated the detection of new reassortant genotypes (e.g., P[6]G9 and P[6]G8) in various parts of the world (14, 15, 23).

Rotavirus G9 strains were first detected as a cause of diarrhea in Philadelphia, Pa., in 1983, where prototype strain WI61 was isolated (10). Two other G9 strains, F45 and AU32, were identified in the 1985-1986 rotavirus season in Japan (49, 52; N. Ikegami, K. Akatani, T. Hosaka, and H. Ushijima, Abstr. VIIth Int. Congr. Virol., p 113, 1987). These three early G9 isolates shared a variety of antigenic and genotypic properties, including membership in the same gene family (Wa genogroup), serotype (P1A[8]G9), E type (long), and subgroup specificity (II), and they had closely related VP7 genes (10, 22, 26, 51, 52, 56). G9 rotavirus strains were also detected in Yugoslavia from 1985 to 1988 (73), in Thailand in 1989 (68), and in India from 1986 to 1993 (15). The isolates from Thailand and India had unusual P types, such as P[19] in Thailand and P[6] and P[11] in India (19, 57). Unlike other G9 strains isolated in this period, the P[11] Indian rotaviruses were detected in newborns with no symptoms of diarrhea. The small number of reports of G9 infections in humans during this time indicated that serotype G9 was relatively rare. Since these initial detections of serotype G9, however, G typing studies have identified G9 rotavirus strains in association with a variety of P types, E types, and subgroup specificities in 23 developing and industrialized countries in recent years, suggesting that this strain has become an important human rotavirus serotype (1-3, 5-7, 11-13, 32, 33, 35, 39, 40, 53, 55, 59, 62-65, 70-72). Sequencing of the VP7 gene of the more recent G9 isolates, identified in both long- and short-E-type isolates, indicated that emerging G9 rotaviruses were probably not direct descendants of the original long-E-type P[8]G9 isolates from the United States and Japan but were closely related to each other (2, 6, 56, 64, 65). The finding that many of the these recent isolates were genotype P[6]G9 with short E types suggested that they may have been formed by reassortment between viruses in different genogroups (38).

Although the segmented nature of the rotavirus genome provides ample opportunities for gene reassortment and the creation of thousands of different genome constellations during mixed infections in vivo, early studies suggested that the G types of most strains were predominantly associated with a single P type, E type, and subgroup specificity (21). This conclusion was supported by RNA-RNA hybridization studies showing that exchange of gene segments between the major gene families represented by long- and short-E-type strains (Wa and DS-1 genogroups) is relatively restricted (47). Of the G serotypes found in the four most common strains worldwide, serotypes G1, G3, and G4 are most often associated with P[8], a long-E-type profile, and subgroup II antigen specificity and belong to the Wa genogroup. In contrast, serotype G2 is associated with P[4], a short-E-type profile, and subgroup I antigen specificity (21). The wide variety of G9 strains that have been isolated suggests that this strain may be unusual in its ability to form reassortants (2, 12, 13, 15, 27, 34, 35, 65, 67, 68, 72). Most notably, five distinct genotypic combinations of G9 strains formed by VP4, VP6, and VP7 gene reassortment between long- and short-E-type isolates were found in a Bangladeshi study, suggesting that intergenogroup reassortment of VP4 and VP7 genes occurred at a high rate (67). It was further proposed that the high rate of mixed infections found in the Bangladeshi study (~23%) may facilitate intergenogroup reassortment of the genes specifying G and P serotype (VP4 and VP7 genes) at a higher frequency in nature than for other common G serotypes (67).

The emergence of serotype G9 as an epidemiologically important strain has raised important issues for rotavirus vaccine development, including whether existing vaccines induce heterotypic protection against the wide variety of circulating G9 strains. Consequently, it will be also important to continue to molecularly characterize G9 strains to develop a better understanding of their diversity and potential for genetic variation, information that may be important in predicting whether these strains may escape immunity elicited by current and planned rotavirus vaccines. One important question concerns the amount of genetic drift that occurs in their neutralizing antigen genes, those for VP4 and VP7, since both are thought to be important in generating immunity and are the targets of vaccines. It may also be important to understand both genetic diversity and potential for reassortment in genes such as those encoding NSP4 and VP6, which have also been implicated as proteins important for virulence and/or protective immunity (4, 8).

In the present study, our objectives were to examine two important issues. To better understand genetic variation present in the VP7 genes of newly emerged G9 strains and study their relationship to strains from the 1980s, we characterized the largest collection of G9 isolates examined to date. Our collection included 40 strains, predominantly from children with gastroenteritis, collected in different geographical locations and from different periods, including long- and short-E-type strains isolated from India in 1993 and 1996 to 1998 and from the United States between 1996 and 2001. Each VP7 gene was characterized by nucleotide sequencing and deduced amino acid analysis of the entire VP7 open reading frame and comparison to other G9 sequences to assess the extent of the variation in the VP7 gene.

Our second objective was to take advantage of this relatively large collection to study the capacity of G9 strains to undergo frequent reassortment in all 11 rotavirus genes. To do this, we examined the level of reassortment occurring among G9 strains from India and the United States and compared the number of different G9 genome constellations circulating in each country. Reassortment in each genome segment was tested by a combination of G and P typing, subgrouping, electropherotyping, and whole-genome hybridization (genogrouping). To try to determine whether high levels of mixed infections present in developing countries affect reassortment frequency, we studied strains from the United States and India, where the rates of mixed infections were 2.1 and 11%, respectively.

MATERIALS AND METHODS

Rotavirus isolates.

Forty human G9 rotavirus strains isolated from stool specimens of patients (both inpatient and outpatient) were used for molecular analysis (Table (Table1).1). Of these, 25 G9 strains were obtained from the collaborating hospital laboratories of the National Rotavirus Strain Surveillance System in the United States between 1996 and 2001, using a protocol described previously (27, 63). The U.S. P[6]G9 and P[8]G9 strains came from one to three cities each for all five rotavirus seasons studied. In addition, the 25 strains represent almost 25% of the G9 strains detected in the United States during those five seasons, and there were representatives from 11 of 12 cities where G9 strains were detected. Ten of the G9 strains were obtained from the stools of inpatients younger than 5 years of age from six different rotavirus surveillance sites in India between 1996 and 1998 (35). The 10 P[6]G9 and P[8]G9 strains came from five of six cities where G9 was found and represented 20% of the G9 strains. The final five strains were obtained in 1993 from stool specimens of neonates born in the nurseries of six hospitals in Delhi, India, who were excreting rotavirus without diarrhea symptoms (9). The strains came from three of the six sites.

TABLE 1.
P type, subgroup, and E type of the G9 strains isolated from patients in the United States and India, 1993 to 2001

Virus cultivation in MA104 cells in roller tube cultures was attempted for strains for which stool specimen was available (30 strains), as described previously (69). Briefly, each stool extract was pretreated with 40 μg of trypsin (Sigma, St. Louis, Mo.) per ml, inoculated onto MA104 cells, maintained in the presence of trypsin (1 to 2 μg/ml), and then harvested 3 to 5 days after infection. After three passages in MA104 cells, cultures were frozen and thawed three times, tested for the presence of antigen by enzyme immunoassay with the Rotaclone rotavirus detection kit (Meridian Diagnostics, Inc., Cincinnati, Ohio), and then passaged in T25 flask cultures of MA104 cells. Twenty-seven of the strains were successfully cultivated and passaged in T25 flasks.

RNA extraction.

Viral RNA was extracted from third-passage viral lysates for the 27 strains that were successfully cultivated in MAl04 cells. A NucliSens extractor (Organon Teknika Corporation, Durham, N.C.) was used to extract the viral RNA after the lysates had first undergone extraction with 1,1,1,2,3,4,4,5,5,5-decafluoropentane (Vertrel XF; Miller Stephenson Chemical Company, Inc., Danbury, Conn.) by using procedures and reagents provided by the manufacturer. Briefly, equal volumes of cell lysates and Vertrel XF were vortexed and then centrifuged. Cell lysates were pipetted into lysis buffer and incubated at room temperature for 10 min. Silica was added to the cell lysate and lysis buffer, and the mixture was added to extractor cartridges. The extractor was run according to the protocol provided, and viral RNA was extracted into 50 μl of elution buffer (Tris HCl, pH 9).

P and G genotypes.

The VP7 (G) and VP4 (P) genotypes of all the strains had been determined in previous studies (9, 27, 35, 63). All strains except those that could not be successfully culture adapted (KC1089, Ph1146, Om46, EM696, SD768, AP13, AC03/4, AC03/5, S18, DV38, S25, N23, and S16) were genotyped again in this study for confirmation and are listed in Table Table1.1. This genotyping was performed with a Qiagen OneStep reverse transcriptase PCR (RT-PCR) kit (Qiagen, Inc., Valencia, Calif.). Briefly, one-round RT-PCR was performed on the RNA extracted from viral lysates by using primers specific for G9 strains, agarose gel electrophoresis, and ethidium bromide staining, as described previously (15, 20, 24).

Subgroup and E-type analysis.

The E types of rotavirus isolates Om526, DV38, BP7, At694, In826, and Ne458 were determined by PAGE and visualized by either ethidium bromide or silver staining (60, 61). Strains De92, Ph158, SD412, SD126, CC149, DL73, DL75, and B41 were subgrouped by enzyme-linked immunosorbent assay, using methods described previously (37). Subgroup antigen specificities could not be assigned to strains S25, S16, S23, and DV28. All other subgrouping and electropherotyping had been done previously and described elsewhere (15, 35, 63).

RT-PCR and automated nucleotide sequencing.

The full-length PCR products of the gene coding for the VP7 protein of all 40 G9 strains were amplified by using a mixture of degenerate Beg9 and End9 primers. Degenerate primers were based on serotypes G1, G2, G3, G4, and G9 VP7 nucleotides 1 to 27 for Beg9 primers and 1036 to 1062 for End9 primers and have been described previously (24, 27). The PCR products were gel purified using a microcentrifuge method with a QIAquick gel extraction kit protocol (Qiagen Inc.). The nucleotide sequence of the entire open reading frame of the VP7 gene was determined by the dideoxynucleotide chain terminator method with a BigDye sequencing kit in an automated sequencer (ABI 377 or ABI 3100; Applied Biosystems Inc., Foster City, Calif.). The primers that were used to completely sequence the gene in both directions included the degenerate Beg9 and End9 primers described previously, as well as internal primers based on previously sequenced G9 VP7 genes (64). Overlapping sequences were analyzed with the Sequencher program, version 4.0.5 (Gene Codes Corp. Inc., Ann Arbor, Mich.).

Genogrouping.

Probes were synthesized against the U.S. and India strains that had been successfully culture adapted, as well as other standard strains for hybridization comparisons, as previously described (47, 48). Briefly, rotavirus RNA was transcribed in vitro in the presence of 32P-labeled GTP. The RNA was denatured for hybridization experiments. The denatured RNA and probes were mixed and allowed to hybridize for 16 h at 65°C and then analyzed by PAGE. Genomic double-stranded RNA was visualized by UV light after ethidium bromide staining. Hybrids between the probe RNA plus strands and the unlabeled minus strands of the individual isolates were visualized by autoradiography and compared with homologous reactions to determine the overall genomic RNA constellation reactions.

Strain BP7 was found to be a mixture of strains, based on the presence of more than 11 bands upon PAGE. Several clones were plaque picked, and at least four combinations of patterns were observed. Since they all looked similar by hybridization, one clone, BP7#2, was chosen as a representative.

Data analysis.

Nucleotide and amino acid comparison indicated that many of the strains had identical or nearly identical (100% deduced amino acid similarity and >99% nucleotide identity) VP7 sequences, so that among the 40 strains, there were only 19 genetically distinct VP7 sequences. Therefore, distance matrices for the VP7 nucleotide and deduced amino acid sequences of 19 representative study strains and reference strains US1205 and AU32 were generated with the Distances program (uncorrected distances method) in the University of Wisconsin Genetics Computer Group (GCG; Madison, Wis.) computer package, version 10.2 (17).

Another distance matrix for the VP7 nucleotide and deduced amino acid sequences of a global collection of G9 strains was generated with the same program. The nucleotide sequences of the VP7 gene of the following G9 strains were obtained from the GenBank sequence database: AU32 (Japan), US1205 (United States), BD431 (Bangladesh), MW47 (Malawi), MC345 (Thailand), Bulumkutu (Nigeria), R160 (Brazil), CIT-254RV (Ireland), t203 (China), 97'SZ37 (China), INL1 (India), 116E (India), 95H115 (Japan), Arg1490 (Argentina), GOS51916/96 (United Kingdom), and RRV.

The pattern of amino acid substitutions between the study strains, reference strains, and the global collection of G9 strains was analyzed with the PRETTY analysis program within the GCG computer package. Phylogenetic relatedness of the VP7 gene of the G9 strains was examined by comparing nucleotide and deduced amino acid sequences between the study strains, reference strains, and a global collection of G9 strains by using the computer software program Clustal X, with 1,000 bootstrap repetitions (66). Phylogenetic trees summarizing the VP7 nucleotide and deduced amino acid sequence relationship between the G9 strains were constructed by the neighbor-joining method with the N-J plot program in the Clustal X package. Phylogenetic analysis with other methods, including the Paupsearch (with 100 bootstrap repetitions) program of the GCG package, with trees generated by using the Treetool program (Michael Maciukenas, University of Illinois) and the Growtree program of the GCG package, gave comparable results. A VP7-encoding gene obtained from strain RRV (serotype G3) was included as an outgroup sequence to better understand the relationship between the G9 rotavirus strains in comparison with non-G9 strains.

Tests for significance were performed using the Simple Interactive Statistical Analysis program on the Web (http://home.clara.net/sisa/t-test.htm). Double-sided P values were used.

Nucleotide sequence accession numbers.

The nucleotide sequences described in this paper have been submitted to the EMBL nucleotide sequence database and are retrievable from GenBank. The accession numbers for the VP7 sequences are as follows: KC244, AJ491174; Ph301, AJ491184; De92, AJ491164; Ph158, AJ491183; SD126, AJ491189; SD412, AJ491190; EM696, AJ491171; At599, AJ491158; Ne413, AJ491178; KC268, AJ491175; KC294, AJ491176; EM39, AJ491169; Ne458, AJ491180; DV28, AJ491167; DV38, AJ491168; B41, AJ491160; N23, AJ491177; S16, AJ491185; S23, AJ491187; AP6, AJ491157; AP13, AJ491154; AC34, AJ491155; AC35, AJ491156; BP7, AJ491161; S18, AJ491186; S25, AJ4911; In364, AJ491172; Om46, AJ491181; Om67, AJ491179; SD768, AJ491191; Om526, AJ491182; EM41, AJ491170; Se121, AJ491192; De18, AJ491163; CC117, AJ491153; CC149, AJ491162; At694, AJ491159; In826, AJ491173; DL73, AJ491165; and DL75, AJ491166.

RESULTS

Rotavirus strains.

We selected 40 G9 isolates from the United States and India that represented the most geographical regions within each country and the greatest range of time periods possible (Table (Table1).1). The strains could be divided into three genetic constellations on the basis of P type, E type, and subgroup specificity, and these constellations were designated A, B, and C. Constellation A (P[6], subgroup I, short E type) included 19 strains isolated from gastroenteritis patients in both the United States and India. Constellation B (P[6], subgroup II, long E type) included seven strains from gastroenteritis patients and Indian neonates excreting rotavirus in the absence of symptoms of diarrhea. Constellation C (P[8], subgroup II, long E type) included 14 strains isolated from gastroenteritis patients in both the United States and India. Strains whose subgroup could not be determined were tentatively assigned to constellations on the basis of other molecular characteristics.

P and G genotypes.

The strains included in this study had been genotyped previously (9, 27, 35, 63). Strains for which the original stool specimens were available were cultivated in MA104 cells, and the P and G genotypes were confirmed by RT-PCR. Thirty of the 40 strains had stool specimens available, and of these, 27 were successfully cultivated and retyped.

VP7 gene sequence comparisons.

Most recently identified G9 strains are more closely related to each other than to G9 strains from the 1980s, such as WI61, F45, AU32, 116E, and MC345 (6, 32, 56, 64, 65). To determine if other recent G9 isolates exhibited similar genetic variation, the VP7 gene sequences of the 40 G9 strains obtained from the United States (n = 25) and India (n = 15) were analyzed and their nucleotide and amino acid sequences were compared with each other and with previously reported sequences retrieved from the GenBank database. Strain AU32, which was isolated in Japan in 1985-1986 was used as the reference strain for the distance matrix (Table (Table2).2). AU32 has a close VP7 gene homology and is almost indistinguishable by genogroup analysis from strains WI61 and F45, isolated during the same period (26, 52, 56).

TABLE 2.
Distance matrix for the VP7 gene of United States and India strains and strain AU32

Among the 40 study strains, 0 to 3.99% amino acid divergence and 0 to 10.04% nucleotide sequence divergence were observed, while 3.68 to 5.83% amino acid divergence and 9.90 to 11.43% nucleotide divergence between the study strains and G9 strain AU32 were found, suggesting that all of the study strains belong to serotype G9. The divergence observed among the study strains would have been even less (0 to 2.45% at the amino acid level and 0 to 2.41% at the nucleotide level) if strains Om46 and Om67, which were highly divergent from the other 38 strains, had been omitted. Thus, most of the VP7 genes of the study strains had nucleotide and deduced amino acid sequences that were either very closely related or virtually identical, including those from strains isolated in different locations and years and having different P types, E types, and subgroup specificities.

Outlier constellation C strains Om46 and Om67, isolated from patients in an Omaha, Nebr., hospital in the 1997-1998 rotavirus season, shared 100% deduced amino acid identity and >99% nucleotide sequence identity. These isolates were unique in that they had relatively low (96.63% amino acid identity and 90.57% nucleotide identity) homology to US1205, a well-characterized constellation A reference strain isolated in the United States during the 1996-1997 season (38). In contrast, all the other strains sequenced were highly homologous to US1205. Om46 had nucleotide and amino acid divergence from strain AU32 similar to its divergence from the recently isolated US1205 (Table (Table22).

Comparison of antigenic regions and amino acid substitutions.

The pattern of amino acid substitutions between strains was examined by using an amino acid alignment with the constellation A strain US1205 VP7 sequence as the reference (Fig. (Fig.1).1). A global collection of G9 strains from the GenBank database that had completely sequenced VP7 genes was also included in the analysis. The data support the hypothesis that the 40 study strains are more similar on average to US1205 than they are to the P1A[8]G9 constellation C strain AU32. The study strains differed from US1205 by an average of 0.30 amino acid substitution per strain in antigenic regions A to D and F, while they differed significantly from AU32 by an average of 6.05 amino acid substitutions (t test; double-sided P value, 0.001).

FIG. 1.
Comparison of deduced amino acid sequences of the antigenic regions of the VP7 gene of the 40 G9 study strains and a global collection of 13 previously studied G9 strains. Sequence alignments were prepared by the GCG program PILEUP, and sequences were ...

Comparison of the P[6] (constellations A and B) and P[8] (constellation C) study strains with US1205 showed significantly more amino acid substitutions per strain among the P[8] strains (0.71 substitution per strain) than the P[6] strains (0.07 amino acid substitution per strain) (t test; double-sided P value = 0.0104), indicating that the P[6] study strains are more closely related to US1205 than are the P[8] study strains.

The 40 study strains were significantly more similar to US1205 than was the global collection of G9 strains, which had an average of 2.75 amino acid substitutions per strain in antigenic regions A to D and F (t test; double-sided P value = 0.0016). Notable exceptions were several recent isolates: Malawian strain MW47 (99.39% amino acid identity overall to US1205 and 100% amino acid identity in antigenic regions), Japanese strain 95H115 (99.39% amino acid identity overall and 100% amino acid identity in antigenic regions), and Brazilian strain R160 (99.08% amino acid identity overall). Some of the study strains shared amino acid substitutions with several global G9 strains. Notably, constellation C strains Om46 and Om67 had I-to-T and T-to-A substitutions at positions 208 and 220 in antigenic region C, and these substitutions were shared by Chinese strain 97SZ37 and Japanese strain AU32.

Phylogenetic analysis.

To better examine the relationship between the entire VP7 gene of the study strains and other full-length G9 sequences in the GenBank database, phylogenetic trees were constructed from their nucleotide and deduced amino acid sequences (Fig. (Fig.2).2). Not unexpectedly, all but two of the U.S. and India study strains grouped together, with some subgroups forming according to genetic constellation, geographical location, and date of isolation. Six other previously characterized G9 strains that were isolated from various regions of the world from 1995 to 1999 and included in this analysis (6, 56, 64, 65) grouped together with the study strains. The two study strains that did not group with the others were the outlier constellation C strains Om46 and Om67. As expected from their substantial nucleotide and amino acid divergence from the other study strains, these strains formed a separate clade with bootstrap values of 1,000 and 996 for the nucleotide and amino acid trees, respectively.

FIG. 2.
Phylogenetic analysis of the VP7 nucleotide sequences of the G9 strains was done with the Clustal X program, with 1,000 bootstrap repetitions. All of the G9 strains were rooted to strain RRV and drawn with the TreeView program. Numbers are bootstrap values, ...

Several other clades distinct from the study strains were formed by G9 strains from the database, including three strains from the 1980s (AU32, 116E, and MC345), two isolates from China, and one each from Ireland and Nigeria (28, 55).

Genogrouping.

We initially examined the genomic RNA relationships among the short-E-type (constellation A) strains as well as their relationships to the DS-1 genogroup, because short-E-type strains usually belong to the DS-1 genogroup. For hybridization, we used a probe prepared from US1205, which was previously shown to belong to the DS-1 genogroup (38). The US1205 probe, a representative of G9 strains with short RNA patterns, formed 11 hybrids with the genomic RNAs from U.S. and Indian short-E-type G9 strains with virtually no aberrantly migrating hybrid bands, regardless of the date or location of isolation, suggesting that short-E-type G9 strains were highly homogeneous (Fig. (Fig.33 and and4).4). The one exception was a slightly aberrant migration pattern for gene 5 (NSP1 gene) of U.S. strain Ph158. Reference strain KUN, a short-E-type G2 strain representative of the DS-1 genogroup, was included in the analysis for comparison, and the KUN genome was found to form eight hybrid bands with the US1205 probe (Fig. (Fig.3).3). Specifically, the US1205 probe did not hybridize to gene 4 or to two genes from the 7/8/9 gene complex of the KUN genome, and a slightly aberrant migration pattern was seen for genes 1, 5, and 10. This suggests that although the short-E-type G9 isolates have a strong relationship with the DS-1 genogroup, divergence away from the prototype DS-1 genogroup isolate KUN has occurred.

FIG. 3.
Patterns of hybridization between RNAs from constellation A and reference rotavirus strains (listed at the top) and a 32P-labeled US1205 RNA probe. (a) Gel stained with ethidium bromide and visualized with UV light; (b) corresponding autoradiogram.
FIG. 4.
Patterns of hybridization between RNAs from constellation B and C and reference rotavirus strains (listed at the top) and a 32P-labeled US1205 RNA probe. (a) Gel stained with ethidium bromide and visualized with UV light; (b) corresponding autoradiogram. ...

The US1205 probe formed one hybrid band with the genomic RNAs from the G9 strains with long RNA patterns and P[8] specificity, including several known members (e.g., AU32) of the Wa genogroup (Fig. (Fig.4).4). This hybrid band most likely represents the G9 VP7 gene shared by US1205 and the G9 strains with long RNA patterns. The US1205 probe formed two hybrid bands with the genomic RNAs from AP6, a P[6]G9 strain with a long RNA pattern. The presence of these hybrid bands is consistent with the sharing of the VP4 and VP7 genes between US1205 and AP6. The US1205 probe formed only one hybrid band with the genomic RNAs from BP7, another P[6]G9 strain having a long RNA pattern, most likely representing the G9 VP7 gene shared by the two strains. Surprisingly, a hybrid band did not form in the region of the VP4 gene, which would have been expected given the sharing of the P[6] VP4 gene between strains BP7 and US1205. This may signify that the BP7 VP4 gene represents a highly divergent P[6] gene, perhaps possessing the same P2C[6] VP4 specificity as that of the supershort human strain AU19 (50). Sequencing of the BP7 VP4 gene will be necessary to confirm this hypothesis.

To determine the genogroup and investigate reassortment among the long-E-type study strains, a total-genome probe made from constellation C strain DL75 was hybridized to constellation B and C study strains. A Japanese long-E-type G9 strain from the Wa genogroup, 95H115, was included in the analysis for comparison because it was an early constellation C isolate possessing a VP7 gene sequence characteristic of recently emerging G9 strains (56). Consistent with the VP7 gene sequencing data, the probe hybridization analysis suggested a greater degree of divergence among the long-E-type strains than the short-E-type strains (Fig. (Fig.5).5). The genomic RNAs from constellation C strains In364, DL73, Om526, De18, Se121, and Em41 all formed 11 bands when hybridized to the DL75 probe. Constellation C strains Om67, CC117, In826, and At694 and reference strain 95H115 formed 10 bands when hybridized to the DL75 probe. Of note were the aberrantly migrating hybrid bands for genes 7/8/9, which code for the VP7, NSP2, and NSP3 proteins, that were formed between the DL75 probe and the genomes of 95H115, In364, and Om67. The DL75 probe formed eight or nine hybrid bands with the constellation B strains AP6 and BP7. It did not form hybrid bands corresponding to gene segment 4, which codes for VP4 and determines P type, with the genomes of AP6 and BP7. This is consistent with the different P types between DL75, AP6, and BP7. An aberrant migration pattern was seen for gene 10 of strains Om67, CC117, In826, AP6, and BP7. Overall, these data clearly demonstrate that the constellation B and constellation C strains belong to the Wa genogroup.

FIG. 5.
Patterns of hybridization between RNAs from constellation C and reference rotavirus strains (listed at the top) and a 32P-labeled DL75 RNA probe. (a) Gel stained with ethidium bromide and visualized with UV light; (b) corresponding autoradiogram.

When probes were made from earlier G9 isolates, reference strains AU32 and 116E, and hybridized with the long-E-type study strains, 6 to 10 hybrid bands were formed, often having aberrant migration patterns (data not shown). These results suggest that although the strains all belong to the Wa genogroup, substantial heterogeneity exists.

DISCUSSION

In the present study, we characterized 40 G9 strains isolated in the United States and India from 1993 to the present to try to gain further insights into genetic variation in the VP7 gene and the ability of serotype G9 strains to generate new strains through reassortment. The identification of a variety of P types, E types, and subgroup specificities associated with serotype G9 in strains isolated from many different locations in the United States and India over a period of 8 years, together with the finding that the VP7 gene of all of these strains has a very high degree of homology, confirms and extends the results of other recent studies (2, 6, 27, 32, 35, 56, 62, 64, 65, 72), suggesting that genome segment reassortment is an important mode of evolution for G9 strains, occurring much more rapidly than evolution by rearrangement or point mutations on individual genes. Our genogrouping analyses support this conclusion and illustrate how reassortment can occur to produce a variety of genome constellations among G9 strains.

In the United States where mixed infections were found in 2.1% of strains during recent years, two common G9 genome constellations have been found, constellation C (P[8]G9, long E type, and subgroup II specificity) and constellation A (P[6]G9, short E type, and subgroup I specificity) (64). A single P[8]G9, short-E-type, subgroup I specificity isolate has also been identified (64). Together with other recent studies, these observations suggest that only two G9 reassortants, constellation A and constellation C, are common in developed countries (3, 7, 27, 32, 56, 70). In India, however, where mixed infections occurred at frequencies of 11% in 1993 (62) and 21% in 1996 to 1998 (35), at least four common G9 genome constellations have been isolated, including constellation C, constellation A, constellation B (P[6]G9, long E type, and subgroup II specificity, and a constellation described as P[11]G9, long E type, and subgroup II specificity. Including the results of other recent studies, seven different G9 constellations have been found in developing countries (2, 57, 65, 67, 72), and at least four of those reassortants are common. Taken together with our data, these results support the hypothesis that the large number of mixed infections in developing countries provides an environment for reassortment to occur, resulting in a degree of reassortment among circulating strains greater than that in developed countries. However, as suggested from other studies and by RNA-RNA hybridization with whole-genome probes in this study, intergenogroup reassortments in genes other than VP4 and VP7 are rare. However, we cannot rule out the possibility that selection bias could have limited our ability to detect such reassortants. In addition, since we analyzed only a small number of G9 strains, the potential for intergenogroup reassortment in genes other than those for VP4 and VP7 should not be discounted. Notably, one large outbreak caused by multisegment intergenogroup reassortants of G2 strains in Australia has been documented (58), demonstrating the potential of this mechanism for generation of rotavirus diversity.

Although reassortment between the major human rotavirus genogroups is thought to be uncommon, reassortment within a genogroup is believed to occur more frequently (46, 48). In agreement with this hypothesis, recent sequencing studies with fragments of each gene segment for both P[8]G1 and P[8]G4 strains isolated in Helsinki, Finland, have provided strong evidence that long-E-type G1 strains undergo frequent intragenogroup reassortment in all segments (42). Other studies have shown that VP7 and VP4 genes of serotypes G1, G3, and G4 and modern G9 strains undergo frequent intragenogroup reassortment, as indicated by the finding that several distinct P[8] VP4 gene lineages are found in association with VP7 genes of more than one serotype (25, 32, 34, 43). Together, these results suggest that the four common long-E-type strains, P[8]G1, P[8]G3, P[8]G4, and P[8]G9, which historically have made up >70% of the circulating rotavirus strains, are continually generating diversity in individual segments through reassortment during mixed infections. The data from the present study support this hypothesis. In general, a large amount of diversity is seen among the long-E-type strains while the short-E-type G9 strains are virtually indistinguishable, regardless of where or when they were isolated. Thus, the finding that great variation exists in the RNA-RNA hybridization profiles among the long-E-type strains, especially with respect to genes 5 and 10, is consistent with the possibility that this variation represents intragenogroup reassortment between genetically distinct long-E-type strains from the Wa genogroup.

The almost complete lack of any variation in RNA-RNA hybridization patterns among the constellation A short-E-type strains suggests that these strains have undergone little, if any, reassortment, consistent with their virtually identical VP7 genes and the postulated recent introduction of these strains into the human population.

Among the long-E-type strains, two study strains, Om46 and Om67, were outliers with respect to both their VP7 gene sequence and their RNA-RNA hybridization profiles. Comparison of the deduced amino acid sequences of the VP7 gene of these strains to reference G9 strains isolated in the 1980s shows them to have several of the same amino acid substitutions as strains AU32, 116E, and MC345, suggesting that they may have a closer evolutionary relationship to these G9 strains than do the other strains in this study. Consistent with this hypothesis, the same substitutions shared by AU32 and the two outlier strains have also been documented to occur in the other two strains isolated in the 1980s, prototype WI61 and the Japanese isolate F45 (56). It may be that Om46 and Om67 represent the distant progeny of the original P[8]G9 clade that has been evolving since the 1980s; alternatively, these strains may have been derived by the independent introduction into the human population of an isolate with a G9 VP7 gene that is not a direct descendant of the isolates from the 1980s.

Several other distinct clades were detected in our evolutionary analysis, demonstrating that a number of distinct G9 lineages in addition to the original lineages from the 1980s and the modern lineage are in circulation. Although these strains are few, they exhibited increased nucleotide and amino acid divergence relative to the majority of the strains characterized, including increased numbers of amino acid substitutions in antigenic regions. Thus, antigenic variation in modern G9 strains may be greater than our current understanding implies, suggesting that additional studies are needed in many countries where few if any strains have been characterized to date.

The high degree of VP7 nucleotide and amino acid sequence homology among all but two of the study strains characterized here, and their great divergence from the earliest G9 strains, supports previous findings suggesting that recent G9 strains are more closely related to each other than to reference G9 strains isolated in the 1980s (32, 56, 64, 65). Such results are consistent with the hypothesis that modern G9 strains were introduced into the population recently by one or more reassortment events. It has been suggested that this reassortment event may have been between the contemporary short-E-type G9 lineage and another G9 strain from a distinct genogroup (38). For the G9 strains that emerged in the United Kingdom beginning in 1996, modern P[6]G9 strains emerged 1 year earlier than modern P[8]G9 rotaviruses. Because P[6]G9 strains emerged first, and it was found that the P[8]G9 strains had distinct VP4 gene lineages characteristic of common P[8]G1, P[8]G3, and P[8]G4 strains that circulate in all countries (25, 34, 43), it was proposed that the emerging P[8]G9 strains evolved through reassortment between common strains and P[6]G9 strains that had been recently introduced into the United Kingdom (32). The observation that the earliest emerging P[6]G9 strains detected in the United States in 1995 had a VP7 gene closely related to those of other modern G9 strains from subsequent years characterized in this and other recent studies (2, 6, 32, 56, 64, 65; V. Jain, H. F. Clark, P. Dennehy, K. Zangwill, C. D. Kirkwood, R. I. Glass, and J. R. Gentsch, Abstr. Am. Soc. Virol. Meeting, abstr. W43-4, 1999) provides further evidence that P[6]G9 strains with short E types could have emerged first by reassortment with an unknown progenitor strain.

The genotype P[6]G9 strains with long E types characterized in this study were first detected in humans during 1993 among Indian neonates born in hospital nurseries, who were excreting rotavirus in the absence of diarrhea symptoms (9). We showed that the VP7 gene of several of the study strains from neonates was virtually identical to those of other study strains from India and the United States, making this the earliest detection of the modern G9 VP7 gene lineage that we are aware of and lending further support for the model that contemporary P[6]G9 strains may have emerged before modern P[8]G9 strains.

The finding of the modern G9 VP7 gene lineage in P[6]G9 rotaviruses isolated from neonates in this study and in another recent study in the United States raises questions about the possible role of rotavirus infections of neonates in the introduction and/or spread of modern G9 strains in the human population. Nosocomial infections of neonates are unusual in that virus is often shed without symptoms of diarrhea and are caused by rotaviruses with P genotypes (e.g., P[6] and P[11]) or P and G genotypes (e.g., P[6]G9 and P[11]G10) that are uncommon in older children with gastroenteritis (16, 19, 29, 31). Since the P[6]G9 strains identified here and in the U.S. neonatal infections had not been found in humans before 1993, it is possible that their unique P-G serotype antigen combination made them particularly well suited to spread and become established in the population due to a lack of protective immunity to the P[6]G9 antigens. In support of this model, P[6]G9 strains with long E types were also first detected in older Indian children with gastroenteritis at about the same time and have subsequently become predominant in India (35, 62). Further reassortment events between P[6]G9 strains and both P[6] and P[8] short- and long-E-type strains could have resulted in the introduction of the G9 VP7 gene into other members of the Wa and DS-1 genogroups, resulting in G9 strains with a variety of genetic constellations capable of producing disease in patients. This model of spread could be further tested through analysis of archival collections from hospital nurseries and hospitalized infants collected in the early 1990s and perhaps the late 1980s, to try to determine whether P[6]G9 strains were first seen in neonates born in nurseries or older children in the community.

Rotavirus surveillance studies carried out in numerous countries throughout the world since 1990 illustrate the increased frequency of detecting serotype G9 strains worldwide (1-3, 5-7, 11-13, 32, 35, 39, 40, 53, 55, 59, 62-65, 70-72). A meta-analysis compiling the data from all the countries shows that serotype G9 had an incidence rate of 5.8%, making it the fourth most prevalent G serotype worldwide during the study period. The increasing detection of serotype G9 suggests that G9 should be considered a fifth common serotype worldwide. If current vaccines against serotypes G1 to G4 fail to protect against serotype G9, an additional G9 reassortant may need to be added to a polyvalent vaccine.

The recent increased detection of G9 strains that were previously considered rare also necessitates continuous worldwide rotavirus surveillance, not only to better determine the prevalence of serotype G9 in different populations but also to detect other unusual strains that might emerge. This surveillance should include retrospective analysis of specimens from previous studies, as well as the employment of accurate diagnostic techniques to characterize strains that are nontypeable by using enzyme-linked immunosorbent assay with monoclonal antibodies alone. Given that G9 has been associated with asymptomatic rotavirus infection in neonates, analysis of only diarrheal fecal specimens may not be sufficient. Surveillance may need to be expanded to include asymptomatic individuals, as they may serve as a reservoir for viral reassortment and evolution of novel G9 strains.

Acknowledgments

This research was supported in part by an appointment to the Emerging Infectious Diseases Fellowship Program administered by the Association of Public Health Laboratories (APHL) and funded by the Centers for Disease Control and Prevention (CDC). In addition, this study was supported in part by a grant-in-aid to T.N. and O.N. from the Japan Society for the Promotion of Science (no. 13670292).

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