Characterization of Nontypeable Rotavirus Strains from the United States: Identification of a New Rotavirus Reassortant (P2A[6],G12) and Rare P3[9] Strains Related to Bovine Rotaviruses

Virology 294, 256–269 (2002) doi:10.1006/viro.2001.1333, available online at http://www.idealibrary.com on

Characterization of Nontypeable Rotavirus Strains from the United States: Identification of a New Rotavirus Reassortant (P2A[6],G12) and Rare P3[9] Strains Related to Bovine Rotaviruses D. D. Griffin,* T. Nakagomi,† Y. Hoshino,‡ O. Nakagomi,† C. D. Kirkwood,* ,§ U. D. Parashar,* R. I. Glass,* J. R. Gentsch,* ,1 and the National Rotavirus Strain Surveillance System 2 *Viral Gastroenteritis Section, Division of Viral and Rickettsial Diseases, Centers for Disease Control and Prevention, U.S. Department of Health and Human Services, Atlanta, Georgia 30333; †Department of Microbiology, Akita University School of Medicine, Akita 010-8543, Japan; ‡Laboratory of Infectious Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland 20892; and §Department of Gastroenterology and Clinical Nutrition, Murdoch Children’s Research Institute, Royal Children’s Hospital, Melbourne, Victoria, Australia Received July 19, 2001; returned to author for revision October 11, 2001; accepted November 28, 2001 Among 1316 rotavirus specimens collected during strain surveillance in the United States from 1996 to 1999, most strains (95%) belonged to the common types (G1 to G4 and G9), while 5% were mixed infections of common serotypes, rare strains, or not completely typeable. In this report, 2 rare (P[9],G3) and 2 partially typeable (P[6],G?; P[9],G?) strains from that study were further characterized. The P[6] strain was virtually indistinguishable by hybridization analysis in 10 of its 11 gene segments with recently isolated P2A[6],G9 strains (e.g., U.S.1205) from the United States, but had a distinct VP7 gene homologous (94.7% aa and 90.2% nt) to the cognate gene from P1B[4],G12 reference strain L26. Thus, this serotype P2A[6],G12 strain represents a previously unrecognized reassortant. Three P3[9] strains were homologous (97.8–98.2% aa) in the VP8 region of VP4 to the P3[9],G3 feline-like reference strain AU-1, but had a high level of genome homology to Italian bovine-like, P3[9],G3 and P3[9],G6 rotavirus strains. Two of the U.S. P3[9] strains were confirmed to be type G3 (97.2–98.2% VP7 aa homology with reference G3 strain AU-1), while the other was most similar to Italian bovine-like strain PA151 (P3[9],G6), sharing 99.0% aa homology in VP7. Cross-neutralization studies confirmed all serotype assignments and represented the first detection of these rotavirus serotypes in the United States. The NSP4 genes of all U.S. P3[9] strains and rotavirus PA151 were most closely related to the bovine and equine branch within the DS-1 lineage, consistent with an animal origin. These results demonstrate that rare strains with P and G serotypes distinct from those of experimental rotavirus vaccines circulate in the United States, making it important to understand whether current vaccine candidates protect against these strains. © 2002 Elsevier Science (USA) Key Words: rotavirus; strain characterization; genotyping; molecular epidemiology; natural reassortants; G12 strains; G6 strains; P3[9] strains; RNA–RNA hybridization; NSP4 gene.

INTRODUCTION

from rotavirus infections each year worldwide (Kapikian et al., 2001). In the United States, approximately 50,000 children are hospitalized due to rotavirus infections, and nearly $1 billion in societal costs can be attributed to rotavirus infections annually. Also, 20–40 infants die from rotavirus disease yearly in the United States (Parashar et al., 1997; Tucker et al., 1998). Good hygiene and sanitation do not prevent the spread of rotavirus infections; thus, vaccines to protect against or lessen the severity of rotavirus disease are being developed. Rotavirus is a triple-layered virus containing 11 gene segments in its inner core. Each of these segments codes for either structural or a nonstructural proteins of which there are six each; gene segment 11 contains two open reading frames (Estes, 2001). The outer capsid is composed of two proteins, VP4 and VP7, that elicit a neutralizing immune response, creating both serotypespecific and cross-reactive immunity. Rotavirus serotype classification is also based on these G (VP7 glycoprotein) and P (VP4 protease-activated) proteins (Estes, 2001). There are eight P and 10 G serotypes of rotavirus

Rotavirus is the most common etiologic agent of severe diarrhea in children. More than 90% of infants have experienced at least one rotavirus infection by 3 years of age. It is estimated that 400,000 to 600,000 children die

1 To whom correspondence and reprint requests should be addressed at Viral Gastroenteritis Section, CDC (Mailstop G04), 1600 Clifton Road NE, Atlanta, GA 30333. Fax: (404) 639-3645. E-mail: [email protected]. 2 Participants in the National Rotavirus Strain Surveillance System include the following: Rebecca Nelson, Arkansas Children’s Hospital, Little Rock; Michelle Hartin, Children’s Hospital of San Diego; Christine Robinson, Children’s Hospital of Denver; Yolanda Arcilla, Medical Center of Delaware, Newark; Gail Bloom, Clarian Health Partners, Indianapolis; David Abel, Children’s Mercy Hospital of Kansas City; Gary Leonardi, Nassau County Medical Center, East Meadow; Paul A. Yam, Children’s Memorial Hospital of Omaha; DeLores Aiazzi, Washoe Medical Center of Reno; H. Fred Clark, Children’s Hospital of Philadelphia; Lisa Rocha, Driscoll Foundation Children’s Hospital of Corpus Christi; Charles Ash, Egleston Children’s Hospital, Atlanta; and Kathy Dugaw, Children’s Hospital and Regional Medical Center, Seattle.

0042-6822/02 $35.00 © 2002 Elsevier Science (USA) All rights reserved.

256

CHARACTERIZATION OF U.S. NONTYPEABLE ROTAVIRUS STRAINS

found in humans (Estes, 2001). The inner capsid of the virus particle is composed of the VP6 protein, which confers group and subgroup antigenicity used to classify rotavirus (Estes, 1996). Group A strains commonly associated with childhood diarrhea may possess subgroup I, subgroup II, both subgroup I and subgroup II, or no subgroup antigens (Kapikian et al., 2001). One nonstructural protein of importance is NSP4, which contains a toxic peptide region, aa 114–135, that has been shown to induce diarrheal disease in neonatal mice (Ball et al., 1996) and has been hypothesized to play a role in the pathogenesis of diarrhea in humans. Attenuation of virus virulence may be associated with aa changes in the toxic peptide region of the NSP4 gene (Zhang et al., 1998). The NSP4 gene can be characterized by nt sequencing into one of four NSP4 gene groups: Wa-like, DS-1-like, AU-1like, and EW-like strains (Ciarlet et al., 2000; Cunliffe et al., 1997; Horie et al., 1999; Kirkwood and Palombo, 1997). Experimental rotavirus vaccines have been designed to provide specific immunity against one or multiple P and G proteins in combination. Such vaccine strategies have necessitated surveillance intensification worldwide to document that the major antigens of experimental vaccines are representative of the globally most common strains. These studies have shown that serotypes G1, G3, and G4 in combination with genotype P[8], and P[4],G2 strains continue to be the four most prevalent rotavirus strains infecting humans (Gentsch et al., 1996; Woods et al., 1992). Serotype combinations P[8],G9 and P[6],G9 appear to be emerging strains that since 1995 have been identified in various parts of the world, including India, the United States, Australia, Bangladesh, Brazil, Italy, the United Kingdom, France, Japan, Ireland, Thailand, Vietnam, the Netherlands, and Malawi (Araujo et al., 2001; Arista et al., 1997; Bon et al., 2000; Cubitt et al., 2000; Cunliffe et al., 1999; Griffin et al., 2000; Maneekarn and Ushijima, 2000; O’Halloran et al., 2000; Oka et al., 2000; Palombo et al., 2000; Ramachandran et al., 1996, 1998; Santos et al., 2001; Unicomb et al., 1999; Van Man et al., 2001; Widdowson et al., 2001; Zhou et al., 2000). Strain pattern prevalences may vary regionally, such as G5s in Brazil, G8s in Brazil, Malawi, Egypt, Finland, and Ghana, and G6s in Italy, Australia, and India (Armah et al., 2001; Cunliffe et al., 1999; Gerna et al., 1990b, 1992; Holmes et al., 1999; Kelkar and Ayachit, 2000; Leite et al., 1996; Palombo and Bishop, 1995; Santos et al., 1998). RNA– RNA hybridization, or genogrouping, has demonstrated that some of the regionally common strains appear to have resulted from in vivo reassortment between animal rotaviruses especially of bovine and porcine origin and human rotaviruses from the three main genogroups, which are designated Wa, DS-1, and AU-1 (Alfieri et al., 1996; Das et al., 1993; Dunn et al., 1993; Iizuka et al., 1994; Nakagomi and Nakagomi, 1993). The U.S. Strain Surveillance System, conducted by the CDC, was initiated in anticipation of the national rotavi-

257

rus vaccination program (Ramachandran et al., 1998). The first licensed vaccine, Rotashield, is a tetravalent vaccine that contains rhesus rotavirus serotype G3 antigens and human rotavirus serotype G1, G2, and G4 antigens (Kapikian et al., 1991). However, because of an association with intussusception, the Rotashield vaccine was suspended from the childhood vaccination schedule, and the vaccine was withdrawn from the market (Centers for Disease Control and Prevention, 1999a,b; Murphy et al., 2001; Nakagomi, 2000). Nonetheless, the emergence of serotype G9 as a possible fifth important G serotype plus increased understanding of regional variation and lack of information on the ability of vaccines to provide cross-reactive immunity further demonstrate the important role of continued rotavirus strain surveillance. Thus, the CDC has continued surveillance in the absence of a commercially available vaccine, but in anticipation of continued vaccine trials with new candidate vaccines, to evaluate rotavirus diversity, the presence of rare strains, strain patterns and prevalence, and the emergence of G9 strains. The surveillance project conducted by the CDC has enlisted 10 to 12 hospitals geographically distributed throughout the continental United States since 1996. During surveillance in the United States from 1996 to 2000, serotype G1 was the predominate strain followed by serotype G2, and the third most common strain was serotype G9 (Griffin et al., 2000). For 4 years, more than 95% of strains were identified as the common serotypes G1, G3, and G4 paired with P[8], genotype combination P[4],G2, and the recurring serotype G9 strains in combination with P[8] or P[6]. The remaining 5% were unusual combinations of common G serotypes and P genotypes, G and/or P mixed infections, rarely identified genotype P[9] strains, and G nontypeables (GNTs). There were two GNTs which could be P genotyped, one as P[9] and one as P[6]. These two GNTs along with the two P[9],G3 strains were cultivated in MA104 cells and analyzed by sequencing, genogrouping, and fluorescence focus reduction neutralization (FFN) for full characterization. This report details the analysis of these unusual strains collected in the United States. RESULTS VP7 sequence analysis Strains Se584 (genotype P[9], long E-type, subgroup I antigens) and Se585 (genotype P[6], short E-type, subgroup I antigens) could not be G serotyped in our routine monoclonal antibody (MAb) serotyping assay or G genotyped using reverse transcription-polymerase chain reaction (RT-PCR) (Griffin et al., 2000). For these two strains, it was necessary to sequence the VP7 gene to determine their G genotypes. For comparison, the VP7 gene of the two strains CC425 and KC814 (P[9],G3, long E-type, subgroup I antigens), identified by MAb enzyme immunoassay (EIA) and RT-PCR as serotype G3, were

258

GRIFFIN ET AL. TABLE 1 Percentage of VP7 nt and aa Homology of Four US Rotavirus Strains to One Another and to Standard Strains G1 to G14 Sequence homology (%) of U.S. virus strains CC425

KC814

Se584

Se585

Rotavirus strain a

G serotype

nt

aa

nt

aa

nt

aa

nt

aa

CC425 KC814 Se584 Se585 Wa S2 AU-1 ST3 OSU PA151 Ty-1 69M WI61 Mc35 YM L26 L338 FI23

3 3 6 12 1 2 3 4 5 6 7 8 9 10 11 12 13 14

100.0 99.5 77.2 74.5 74.7 73.7 91.3 74.2 77.1 78.9 64.9 75.6 79.4 75.8 77.1 75.5 76.0 79.7

100.0 99.0 90.0 89.2 87.7 81.9 97.2 83.1 88.3 90.2 70.6 89.0 90.2 88.3 90.2 86.5 86.2 88.7

— 100.0 77.5 74.8 74.7 73.9 91.8 74.4 77.3 79.0 65.3 75.9 79.8 76.0 77.2 75.8 76.5 79.9

— 100.0 90.3 89.2 87.7 81.6 98.2 83.1 88.7 90.5 70.9 88.7 90.8 88.7 90.2 87.4 86.5 89.6

— — 100.0 73.6 72.5 73.2 76.0 74.7 75.8 96.3 65.3 73.0 74.2 74.3 75.6 73.2 74.2 73.1

— — 100.0 85.2 84.6 80.9 89.6 85.6 87.6 99.0 71.9 87.3 87.6 87.3 88.6 83.6 84.6 86.6

— — — 100.0 74.5 72.2 74.5 72.3 75.1 74.5 64.3 72.6 76.3 79.1 73.4 90.2 75.1 74.9

— — — 100.0 84.2 81.4 88.2 81.4 86.1 86.1 67.7 84.2 88.2 84.2 86.1 94.7 84.8 84.5

Note. Boldface type indicates high strain homology typically shared among strains of the same G serotype. a The VP7 sequences were obtained from published reports and the GenBank database by accession number: Wa (K02033), S2 (M11164), AU-1 (D86271), ST3 (X13603), OSU (X04613), PA151 (L20881), Ty-1 (L01098), 69M (Green et al., 1989), WI61 (Green et al., 1989), Mc35 (D14033), YM (M23194), L26 (M58290), L338 (D00843), FI23 (M61876).

also sequenced. Partial nt and deduced aa sequences of the VP7 gene for each of these strains were obtained: Se584 (908 nt, 301 aa), Se585 (970 nt, 326 aa), CC425 (1009 nt, 326 aa), and KC814 (1007 nt, 326 aa). This analysis showed that strain Se584 is most closely related to strain PA151, a serotype G6 strain isolated in Italy, sharing 96.3% nt and 99.0% aa homology, whereas strain Se585 is most similar to strain L26, a serotype G12 strain identified in the Phillippines, sharing 90.2% nt and 94.7% aa homology (Table 1). The G3 strains CC425 and KC814 are extremely homologous in their VP7 nt (99.5%) and aa (99.0%) sequences to each other and exhibited the most similarity to the G3 reference strain AU-1 (91% nt, 97–98% aa), an isolate from Japan. The VP7 antigenic regions (A, B, C, and F) of these strains were compared with those of strains having the same G genotype (data not shown). Se584 demonstrated 1 aa substitution in each antigenic region, A, B, and C, when compared with human serotype G6 strain PA151, whereas 2 to 4 aa substitutions were seen in all antigenic regions, A, B, C, and F, when compared with serotype G6 bovine strain NCDV. Strain Se585 exhibited an identical antigenic B region when compared with serotype G12 reference strain L26 together with 1 or 2 aa substitutions in antigenic regions A, C, and F. In contrast, the two G3 strains CC425 and KC814 shared identical antigenic regions A, B, and F and had only 1 aa substi-

tution in region C when compared to the VP7 gene sequence of strain AU-1. VP4 sequence analysis The P genotypes of strains Se584, Se585, CC425, and KC814 were identified by RT-PCR. To confirm the P genotype assignment of these strains and to compare their relationships to other human rotavirus P serotypes and genotypes, sequence analysis of the VP8 portion of the VP4 gene was carried out. The strains were compared with one another and with standard human strains of various P genotypes [4, 6, 8, 9, 10, 14] (Table 2). The U.S. P[9] strains (Se584, CC425, and KC814) share approximately 96–98% nt and aa homology with strain P3[9] AU-1. The P[6] genotype of strain Se585 was confirmed when it was demonstrated to have 95% nt and aa identity with strain ST-3, P2A[6],G4. It also shares 99.3% VP4 nt and aa homology with strain US1205, a P2A[6],G9 isolated in the United States in 1997 (data not shown). This close relationship was also reflected in the phylogenetic analysis wherein Se585 clustered tightly with US1205 and other recent isolates possessing P[6] specificity (Fig. 1). NSP4 sequence analysis The potential role of NSP4 in virulence makes it an important gene to study; therefore the NSP4 nt and

CHARACTERIZATION OF U.S. NONTYPEABLE ROTAVIRUS STRAINS

259

TABLE 2 Sequence Comparison of the VP4 Gene of U.S. Rotavirus Strains with the Corresponding Sequences of Standard Rotavirus Strains Sequence homology (%) of U.S. virus strains CC425

KC814

Se584

Se585

Strain a

P genotype

nt

aa

nt

aa

nt

aa

nt

aa

CC425 KC814 Se584 Se585 DS-1 ST3 Wa AU-1 69M Mc35

[9] [9] [9] [6] [4] [6] [8] [9] [10] [14]

100 99.5 98.3 60.9 60.2 59.8 59.6 97.4 62.4 77.7

100 99.6 99.6 62.4 62.8 61.7 62.5 98.2 67.3 89.9

— 100 98.6 61.4 60.5 60.7 60.6 96.6 62.6 77.6

— 100 99.3 62.3 63.1 62.4 63.5 97.9 67.5 89.8

— — 100 60.7 60.5 59.7 60.0 96.5 62.2 77.6

— — 100 62.4 63.0 61.9 62.3 97.8 67.7 89.6

— — — 100 68.7 95.2 71.4 60.9 64.6 58.5

— — — 100 75.0 96.7 76.1 62.7 67.8 61.6

Note. Boldface type indicates high strain homology typically shared among strains of the same P genotype. a VP4 sequences from standard strains were obtained from the GenBank database using accession numbers DS-1, P11196; ST3, L33895; Wa, M96825; AU-1, D10971; 69M, M60600; Mc35, D14032.

deduced aa sequences were analyzed for strains Se584, Se585, CC425, and KC814. In addition, the NSP4 sequences for strains PA151 and L26 were determined in this report. In contrast to their AU-1-like VP8 gene fragments, the NSP4 genes of the U.S. P[9] strains (Se584, CC425, and KC814) and PA151 segregated with the DS-1 lineage by phylogenetic analysis (Fig. 2). However, there are several distinct sublineages supported by bootstrap analysis among DS-1-like NSP4 genes, including one represented by U.S. P[9] strains CC425, KC814, and Se584 and a second represented by equine strains H2, FI23, and FI14 (Fig. 2). The deduced aa sequences of the NSP4 gene of the U.S. P[9] strains CC425 and KC814 were identical to each other and shared 99.4% identity with strain Se584. These strains were more closely related to the NSP4 gene of PA151 and equine and bovine strains including NCDV (96.0 to 97.1% aa identity) than to other NSP4 genes within the DS-1 lineage (94.3–95.4%) (data not shown). Consistent with the phylogenetic analysis, these two sublineages were characterized by 3–6 aa substitutions conserved within the group but not present in other members of the DS-1 lineage (data not shown). The U.S. P[6],G12 strain Se585 belonged to the third and largest DS-1-like NSP4 gene sublineage which included other recent isolates, such as US1205 and US1250 (P2A[6],G9), and G2 reference strains such as S2 and KUN. The NSP4 gene of strain L26 appears to be most like the NSP4 gene of strain 1076 (P2A[6],G2), which is also found in the DS-1-like cluster. Antigenic characterization Two of the three P[9] strains, CC425 and KC814, were identified originally as serotype G3 by MAb EIA that we routinely use for serotyping our rotavirus positive speci-

mens. The third P[9] strain, Se584, was closely related to serotype G6 by sequence analysis and confirmed as serotype G6 by reactivity with G6 MAb IC-3 utilized in our standard EIA format. We were unable to obtain a G serotype for strain Se585 by our routine MAb EIA. This was not unexpected since sequencing demonstrated this strain to belong to genotype G12 and we do not have a MAb with this specificity. The three long electropherotype P[9] strains and the short electropherotype P[6] strain possessed subgroup I antigens. To confirm the P and G serotypes, FFN was performed on cultivated strains CC425, Se584, and Se585 using hyperimmune antisera raised against 16 strains representing 14 distinct G serotypes (data not shown). Strain CC425 was neutralized by hyperimmune antisera to serotype G3 strains P and RRV to titers of 10,240 and 40,960, respectively. Antisera to serotype G6 strains UK and NCDV neutralized strain Se584 to titers of 40,960 and 10,240, respectively. The G12 serotype-specificity of Se585 was confirmed when it was neutralized to a titer of 10,240 by antisera created against L26. The P serotypes of strains CC425 and Se584 were confirmed to be P3[9] when these strains were neutralized by antisera raised against reassortants PA151xDS-1 and K8xDS-1 to titers of 10,240 and 2560, respectively (data not shown). Antisera against reassortant ST3xDS-1 neutralized strain Se585 to a titer of 1280, confirming that this strain belongs to serotype P2A[6]. Together these results demonstrate that the complete serotype designations of these strains are CC425, P3[9],G3; Se584, P3[9],G6; and Se585, P2A[6],G12. The finding that CC425 belongs to serotype P3[9] strongly suggests that KC814 possesses the same P serotype since the deduced aa

260

GRIFFIN ET AL.

FIG. 1. Phylogenetic tree of VP4 deduced amino acid sequences. The tree was constructed using the PaupSearch Program of the Genetics Computer Group (GCG) Sequence Analysis Software Suite (Devereux et al., 1984) using the neighbor-joining method and a multiple sequence format file constructed with the PILEUP program of GCG as input. The distance between aa sequences is expressed as the number of aa substitutions per site and is proportional to the horizontal distance between sequences. Only bootstrap probabilities greater than 90 out of 100 are given. An asterisk denotes U.S. strains sequenced in this report. The following VP4 sequences were taken from the GenBank database (accession numbers): Mc35 (D14032), AU-1 (D10971), AU720 (D14618), RO5193 (X90733), K8 (D90260), RO6460 (X90736), MZ58 (D14622), PA151 (D14623), PCP5 (D14624), FRV1 (D10970), AU228 (D14615), RO5829 (X90734), Cat2 (D13403), 02-92 (AB008289), US1205 (AF079356), MW23 (AJ278253), GR630/86 (AF161825), HN221 (L20878), M37 (L20877), VE7156 (L25265), NNB1 (AF076926), ST-3 (L33895), 1076 (M88480), AU19 (AB017917), Gottfried (M33516), and 4F (L10359). G?, we were unable to find a reference to the G type for strain HN221.

homology between the VP8 regions of the two strains is 99.6%. Genogrouping The U.S. P3[9] strains had either serotype G3 or G6 specificity and a VP8 region closely related to AU-1, raising the possibility that these strains could be closely related to the feline rotavirus-like AU-1 reference strain. However, previously described strains with these serotypes from Italy (serotype P3[9],G3 and P3[9],G6) have been shown to be reassortants between an AU-1-like strain and a bovine-like strain. To determine whether the U.S. P3[9] strains would show similar relationships, a total genome probe produced from AU-1 was used for genogrouping experiments. When the AU-1 probe was hybridized with the U.S. P3[9] strains CC425, KC814, and Se584, only four, four, and three bands were formed, respectively (Fig. 3). This result suggested that these

strains were reassortants between the AU-1 genogroup and another genetic lineage, such as the bovine-like P3[9] strains isolated in Italy that gave a similar hybridization pattern with AU-1 probes (Iizuka et al., 1994; Nakagomi et al., 1992a). To test this, we made probes to bovine strain NCDV, which previously had reacted strongly with P[9],G3 and P[9],G6 strains from Italy. As we hypothesized, the NCDV probe was more homologous to the U.S. P3[9] strains, forming five or six strong hybrid bands and one to three intermediate or weak bands with each strain (seven bands with CC425 and KC814 and eight bands with Se584). Total genome probes made from U.S. strains CC425, Se584, and KC814 were hybridized to AU-1, bovine NCDV, and representative Italian bovine-like P[9],G3 (PCP5) and P[9],G6 (PA151) strains (Fig. 4; data from KC814 probe experiments are not shown). As predicted, the CC425 and KC814 probes formed four bands with AU-1 while the

CHARACTERIZATION OF U.S. NONTYPEABLE ROTAVIRUS STRAINS

261

FIG. 2. Phylogenetic tree of NSP4 deduced amino acid sequences. The tree was constructed using the PaupSearch program of the Genetics Computer Group (GCG) Sequence Analysis Software Suite (Devereux et al., 1984) using the neighbor-joining method and a multiple sequence format file constructed with the PILEUP program of GCG as input. The distance between aa sequences is expressed as the number of aa substitutions per site and is proportional to the horizontal distance between sequences. Only bootstrap probabilities greater than 90 out of 100 are given. An asterisk indicates U.S. strains sequenced in this report. The following NSP4 sequences were taken from the GenBank database (accession numbers): EHP (U96336), EC (U96337), EW (AB003805), Wa (K02032), ST-3 (U59110), VA70 (U83798), H1 (AF144800), OSU (D88831), YM (X69485), 116E (U78558), RV-3 (U42628), M37 (U59109), AU32 (D88830), US1099 (AJ236759), US1205 (AF079358), US1250 (AJ236756), KUN (D88829), SA11 (K01138), UK (K03384), RV-5 (U59103), 1076 (U59105), NCDV (X06806), H2 (AF144801), FI23 (AF144802), FI14 (AF144803), FRV-1 (D89874), AU-1 (D89873), R2 (AB005472), RRV (L41247), FRV64 (D88833), and RS15 (D88832). The P serotype of strain R-2 may be P11? (Estes, 2001).

Se584 probe formed three bands with AU-1, and all three probes from the U.S. strains formed eight bands with NCDV. These strains exhibited significant homology with the Italian bovine-like strains, sharing at least seven or eight gene segments with each. No significant hybrid bands were formed between the U.S. P3[9] strains and standard human strains DS-1 and Wa (data not shown). Genogrouping of Se585 (P2A[6],G12) was also conducted. As expected from its genotypic properties, Se585 shares 10 of its 11 gene segments with U.S. DS-1 genogroup strain US1205, a P2A[6],G9 isolated in 1997, (Fig. 5) and 7 gene segments with G12 strain L26 (data not shown). Both Se585 and US1205 share multiple bands with strain DS-1 (data not shown). Few or no hybrid bands were seen when the Se585 probe was hybridized with representative strains of NCDV, OSU, AU-1, and Wa.

DISCUSSION Rotavirus continues to be a devastating disease among children, and the best hope for its prevention is the development of an effective vaccine. Rotavirus surveillance is a necessary step in designing vaccines and vaccine strategies to identify regional strain patterns and potential emerging strains. During routine rotavirus surveillance in the United States, a novel reassortant P2A[6],G12, having a VP7 gene not previously seen in a short E-type strain, and several rare P3[9],G6 and P3[9],G3 isolates, which have not been previously isolated in the United States, were found (Table 3). Because of potential implications for rotavirus vaccine programs, these unusual strains were characterized in detail by the use of MAb EIA, RT-PCR, sequencing of the VP4, VP7, and NSP4 genes, FFN, and RNA–RNA hybridization. Se-

262

GRIFFIN ET AL.

FIG. 3. Hybridization patterns between RNAs from rotavirus strains listed at the top and RNA 32P-labeled probes listed at the bottom. The top gel (a) has been stained with ethidium bromide and visualized with UV light. The bottom gel (b) is the corresponding autoradiogram.

rotype G12 has been documented only once previously (in the Philippines) in children with diarrhea. The reference G12 strain, L26, belonged to serotype P1B[4],G12 and had SG I antigens and a long E type (Taniguchi et al., 1990). On the other hand, P3[9] strains are more widely distributed. These strains have been identified in a vari-

ety of countries including Japan, Malaysia, Brazil, Venezuela, Italy, Israel, South Africa, Guinea-Bissau, and now in the United States (Fischer et al., 2000; Gerna et al., 1990a, 1992; Griffin et al., 2000; Leite et al., 1996; Nakagomi et al., 1987; Rasool et al., 1993; Santos et al., 2001; Shif et al., 1994; Steele et al., 1993). Serotype G6 strains were originally isolated from diarrheic calves and were thought to be unique to the bovine species (Kapikian and Chanock, 1990; Snodgrass et al., 1990). Later, G6 strains were detected in humans in Italy, Australia, and India (Gerna et al., 1992; Kelkar and Ayachit, 2000; Palombo and Bishop, 1995). The detection and characterization of these U.S. G6 strains have broadened our knowledge of their global distribution and have contributed to our understanding of the diversity of strains circulating in the United States. Since rotavirus has 11 gene segments, the theoretical possibility of reassortment or exchange of gene segments between two or more strains during a mixed infection is great. However, in early studies, natural reassortants between, for example, subgroup I strains (usually G2 with short electropherotypes) and subgroup II strains (usually G1, G3, or G4 with long electropherotype) were rarely detected by RNA–RNA hybridization experiments, suggesting that rotavirus genes are genetically linked (Nakagomi et al., 1987). In addition, reassortment between subgroup I and subgroup II strains could be obtained in vitro only at low frequency, which supported the hypothesis that rotavirus genes are linked in some cases (Garbarg-Chenon et al., 1984). Studies using RNA–RNA hybridization assays applied to larger numbers of human rotavirus strains have in general supported these early findings (Flores et al., 1982; Nakagomi and Nakagomi, 1993). Using radioactive or digoxigenin-labeled total genome probes synthesized by in vitro transcription or cDNA synthesis, the genetic relatedness of RNAs from new strains can be compared with RNA from reference strains on the basis of the number of visible bands formed after hybridization with the desired probes. An increasing number of hybrid bands and comigration of hybrid bands of test strains with homologous hybrids indicate a higher level of genetic homology (Nakagomi and Nakagomi, 1991; Nakagomi et al., 1992b; Ward et al., 1990). Most human rotaviruses can be divided into one of two major genogroups, Wa or DS-1, or one minor genogroup, AU-1, on the basis of overall genomic RNA homology (Nakagomi et al., 1989). Reassortants between genogroups have been described occasionally; however, in some regions, intergenogroup reassortment may occur at high frequency (Nakagomi and Nakagomi, 1991; Nakagomi et al., 1992b; Palombo et al., 1996; Ramachandran et al., 2000; Unicomb et al., 1999; Ward et al., 1990). In addition, several rotavirus reassortants have been described that have been derived from strains of human and animal origin. This mechanism appears to be important for gen-

CHARACTERIZATION OF U.S. NONTYPEABLE ROTAVIRUS STRAINS

263

FIG. 4. Hybridization patterns between RNAs from rotavirus strains listed at the top and RNA 32P-labeled probes listed at the bottom. The top gel (a) has been stained with ethidium bromide and visualized with UV light. The bottom gel (b) is the corresponding autoradiogram.

eration of genetic diversity in rotaviruses (Alfieri et al., 1996; Browning et al., 1992; Das et al., 1993; Holmes et al., 1999; Urasawa et al., 1993).

Strain Se585 (P2A[6],G12) appears to be a naturally occurring reassortant between a serotype G12 strain such as L26 (P1B[4],G12) and a strain closely related to

264

GRIFFIN ET AL.

FIG. 5. Hybridization patterns between RNAs from rotavirus strains listed at the top and RNA 32P-labeled probes listed at the bottom. The top gel (a) has been stained with ethidium bromide and visualized with UV light. The bottom gel (b) is the corresponding autoradiogram.

the DS-1 genogroup isolate US1205 (P2A[6],G9) (Kirkwood et al., 1999). The VP7 gene sequence of Se585 exhibited the most similarity to the VP7 gene sequence of strain L26 and was neutralized by antisera against strain L26. The VP4 gene sequence of strain Se585 was highly homologous with the VP4 gene sequence of strain

US1205. When these two P2A[6] strains were compared in a phylogenetic tree with other representative P[6] strains, they segregated with recently described reassortant MW23 (P2A[6],G8) from Malawian children and human South African strain GR630 (P2A[6],G4) within the P2A[6] subtype cluster (Cunliffe et al., 2000; Pager et al., 2000). Genogroup analysis demonstrated that Se585 and US1205 are virtually indistinguishable with the exception of the VP7 gene. Thus, both the relatively common short electropherotype G9 strains isolated over the past several years in the United States and perhaps elsewhere (Ramachandran et al., 2000; Unicomb et al., 1999) and the rare G12 strain appear to be single-segment (VP7 gene) reassortants of a closely related parental virus from the DS-1 genogroup. The G serotype of the short E-type parent and the strains that donated the VP7 genes are not known. Nonetheless, these studies suggest that the short electropherotype profile strain may have an unusual propensity for generating new serotype combinations by VP7-gene and/or VP4-gene replacement (Cunliffe et al., 2000; Ramachandran et al., 2000). Similar mechanisms have been proposed for the generation of the highly prevalent short electropherotype P2A[6],G8 strains from Malawi (Cunliffe et al., 2000). The P3[9] strains described here are genetically more similar to the P3[9] strains isolated in Italy than to the AU-1-like P3[9] strains characterized in Japan (Gerna et al., 1990a, 1992). The P3[9],G3 strains PCP5 and PA151 collected in Italy were demonstrated by genogrouping to be reassortants between bovine and AU-1-like strains (Iizuka et al., 1994; Nakagomi et al., 1992a). Interestingly, the genomes of the U.S. P3[9] strains were more homologous with Italian P3[9] strains than with other reference strains, and their homology with strains NCDV and AU-1 was similar to that reported for the Italian P3[9] strains (Iizuka et al., 1994; Nakagomi et al., 1992a). Previous reports suggest that P3[9] strains can be further classified into three subgenogroups on the basis of RNA–RNA hybridization and VP8 sequence homology. As shown by Gollop et al. (1998), using Israeli strains RO5193, RO6460, and RO5829, strains that are more genetically similar, sharing 11 of 11 gene segments with AU-1, tend to share higher VP8 sequence similarity. This VP8 sequence identity decreases as the number of hybrid bands shared with AU-1 declines (Gollop et al., 1998). We did not find this association in our VP8 sequence analysis. These three U.S. P3[9] strains did form a separate VP4 cluster among the P3[9] strains even though they shared 96– 98% nt and aa homology with the AU-1 strain, confirming and extending an observation that the P3[9] gene is highly homogeneous irrespective of the year and place of isolation or of the host species (Isegawa et al., 1992; Nakagomi et al., 1993). The NSP4 genes of P3[9] strains CC425, KC814, Se584, and PA151 do not segregate into the AU-1-like NSP4 group but, consistent with the relationships of these

CHARACTERIZATION OF U.S. NONTYPEABLE ROTAVIRUS STRAINS

265

TABLE 3 Summary of Properties of U.S. Strains Se584, Se585, KC814, and CC425 Characteristic (gene segment encoded by)

Strain

P serotype (4)

G serotype (7, 8, or 9)

E type (11)

SG (6)

CC425 KC814 Se584 Se585

P3[9] P3[9] P3[9] P2A[6]

3 3 6 12

Long Long Long Short

I I I I

strains to animal rotaviruses by genogrouping, are most closely related to equine and bovine strains in the DS1-like NSP4 group. The NSP4 genes of the U.S. P3[9] strains and the equine strains clustered in distinct sublineages of the DS-1 NSP4 gene lineage. Also, there were no remarkable changes in the toxic peptide region among the U.S. P3[9] strains and AU-1 and NCDV. The strains described in this report caused severe illness in children ages 1–8 years old (two P3[9] strains were from 8- and 7-year-old children, respectively, while the third P3[9] strain and the G12 isolate were from 1-year-old children) who sought medical attention at a hospital. It is unusual that children over 5 years old contract and develop serious rotavirus disease since more than 90% of children less than 3 years old have had at least one rotavirus infection, which often protects against serious disease during the second infection (Kapikian et al., 1996). The mean and median ages of these four children (4.25 and 4 years, respectively) are older than the overall study set (1.5 years and 1 year, respectively). Thus, it is interesting to speculate that the P3[9],G3 and P3[9],G6 strains caused secondary infections in these older children because one or both of the VP4 and VP7 neutralization antigens were distinct from those of common human rotaviruses, allowing escape from immunity and subsequent disease. Studies with larger numbers of rare and novel strains will be necessary to assess the significance of this difference. Serotype G12 has been identified only once before in humans from the Philippines and was identified for the first time in the United States (Taniguchi et al., 1990). Strain WI61, P[8],G9, was first detected in the United States approximately 15 years ago and was not identified again until 1995 (Clark et al., 1987); however, serotype G9 now represents the third most common G serotype in this country (Griffin et al., 2000; Ramachandran et al., 1998). It will be important to continue to monitor rotavirus strains in the future to detect emerging strains and to determine effective vaccine strategies. MATERIALS AND METHODS Viruses Rare strains were isolated from stool specimens collected at hospital laboratories participating in the Na-

Genogroup (1–11) Human Human Human Human

AU-1/bovine AU-1/bovine AU/bovine DS-1

tional Rotavirus Surveillance System established by the CDC in 1996 (Griffin et al., 2000; Ramachandran et al., 1998). Strain CC425 was identified during the 1997–1998 rotavirus season in Texas, and strains KC814, Se584, and Se585 were isolated in 1998–1999 in the states of Missouri and Washington. Each specimen was prepared as a 10% stool/PBS extract after clarification with Freon. Samples were screened by EIA (Rotaclone, Meridian Diagnostics) for the presence of rotavirus. Of these, 1316 positive specimens subsequently were subjected to Gtyping by EIA and/or RT-PCR and P-typing by RT-PCR. G-nontypeables were amplified by RT-PCR for nt sequencing. These novel strains were activated with trypsin and passaged three times in roller tubes of monkey kidney cells (MA104) and two times in stationary flasks. All passages were cultivated with 1 ␮g/ml trypsin in the maintenance medium. For genogrouping and neutralization studies, the viruses were also plaque-purified and further passaged before testing. The origins of reference strains Wa, DS-1, P, AU-1, RRV, ST-3, OSU, UK, NCDV, PA151, PCP5, Ch2, 69M, WI61, B223, YM, L26, L338, and FI23 have been described previously (Arias et al., 1989; Bohl et al., 1984; Browning et al., 1991a,b; Clark et al., 1987; Gerna et al., 1990a, 1992; Matsuno et al., 1985; McNulty et al., 1979; Mebus et al., 1971; Nakagomi et al., 1987; Piec and Palombo, 1997; Stuker et al., 1980; Urasawa et al., 1990; Wen et al., 1995; Woode et al., 1975, 1983; Wyatt et al., 1983, 1984). Serotyping EIA To G-serotype rotavirus strains, MAbs KU-4 (G1), 5E8 (G1), IC10 (G2), S2-SG10 (G2), YO-1E2 (G3), G3-159 (G3), ST-2G7 (G4), and F45:8 (G9) were utilized in a modified assay previously described (Coulson et al., 1987; Taniguchi et al., 1987). Subgrouping was performed, using MAbs 255/60 (subgroup I) and 631/9 (subgroup II), simultaneously with G-serotyping (Greenberg et al., 1983). To serotype strain Se584, MAb IC3 (G6) was incorporated into our standard assay (Snodgrass et al., 1990). A serotype or subgroup was assigned by a twofold higher optical density value against the homologous MAb com-

266

GRIFFIN ET AL.

pared to the heterologous MAb reactions (Taniguchi et al., 1987). Electropherotypes RNA was extracted from fecal specimens by the glass powder method or from cell lysates by phenol–chloroform followed by the glass powder method (Gentsch et al., 1992). To examine RNA profiles of viruses, RNA extracts were electrophoresed on 10% polyacrylamide gels. Silver staining was applied to visualize bands as previously described (Merril et al., 1981). RT-PCR Rotavirus dsRNA was extracted from stool specimens by use of the glass powder method in the presence of 4 M guanidine thiocyanate and from infected MA104 cells by use of phenol–chloroform and precipitation by ethanol followed by the glass powder procedure (Gentsch et al., 1992). Identification of G and P types for some strains was done utilizing primers in a seminested RT-PCR (Das et al., 1994; Gentsch et al., 1992; Gouvea et al., 1990). The VP8 section of the VP4 gene was amplified using the consensus primers Con2 and Con3 (Gentsch et al., 1992). To amplify the VP7 gene, degenerate beg9 and end9 primers were used (Gouvea et al., 1990). A reaction contained 10 pmol each of beg9 homologous to strains Wa and S2 VP7 nt 1–27 and 4 pmol each of end9 complementary to nt 1036–1062 of reference strains Wa (G1), S2 (G2), and ST3 (G4) and recent isolates 92A (G2, Accession No. U73947) and 95-91 (G3, Accession No. D86266) (Piec and Palombo, 1997; Wen et al., 1995). Nucleotide and amino acid sequencing The QIAquick Gel Extraction Kit (Qiagen, Chatsworth, CA) procedure was used to purify 1.2% SeaKem GTG agarose gel (FMC Bioproducts, Rockland, ME) RT-PCR products stained with ethidium bromide. DNA sequencing was performed on an automated sequencer (Applied Biosystems Model 377), using the dideoxynucleotide chain termination method and the Prism Ready Big Dye Terminator Cycle Sequencing Kit (PE Applied Biosystems, Foster City, CA). Internal primers were synthesized for VP4 and VP7 genes on the basis of sequences obtained from the original consensus primers. These oligonucleotide primers were synthesized in the CDC Biotechnology Core Facility. Overlapping sequence fragments were analyzed manually using the Sequencher program (Gene Codes Corp., Inc., Ann Arbor, MI) and subsequently compared using the PILEUP and distances programs of the University of Wisconsin Genetics Computer Group software suite (Devereux et al., 1984). The new nucleotide sequences described in this paper have been submitted to the EMBL Nucleotide Sequence Database. The accession numbers for the NSP4 nucleotide sequences of strains L26 and PA151 are

AJ311732 and AJ311733, respectively. The accession numbers of the VP7, VP4, and NSP4 nucleotide sequences follow: for strain CC425, they are AJ311738, AJ311734, and AJ311728, respectively; for strain KC814, they are AJ311739, AJ311735, and AJ311729, respectively; for strain Se584, they are AJ311740, AJ311736, and AJ311730, respectively; and for strain Se585, they are AJ311741, AJ311737, and AJ311731, respectively. FFN FFN was incorporated to characterize the antigenic VP4 and VP7 phenotypes of the tissue culture-adapted strains as previously described (Coulson et al., 1985; Hoshino et al., 1984). The reciprocal of the dilution giving a 60% reduction in the number of infected cells compared with that in the virus control wells was used to express the neutralizing antibody titer. The preparation and specificity of antisera to reassortant strains WaxDS-1, S2xUK, DS-1xRRV, ST3xDS-1, PA151xDS-1, K8xDS-1, K8xDS-1, and 69MxDS-1 and to reference strains belonging to serotypes G1 to G14 have been described previously (Hoshino and Kapikian, 1996; Hoshino et al., 1984). Genogroup analysis Probes were synthesized against the novel U.S. tissue culture-adapted strains and other standard strains for hybridization comparisons as previously described (Nakagomi and Nakagomi, 1993; Nakagomi et al., 1989). In brief, 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 polyacrylamide gel electrophoresis. Genomic dsRNA 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. ACKNOWLEDGMENTS This work was supported in part by a grant from the National Vaccine Program Office of the CDC. We thank John O’Connor for editorial assistance and Drs. Ruth Bishop, Barbara Coulson, Harry Greenberg, Shozo Urasawa, and Koki Taniguchi for monoclonal antibodies. D. D. Griffin was supported by a grant from the Oak Ridge Institute for Science and Education, U.S. Department of Energy. C. D. Kirkwood was supported by the Atlanta Research and Education Foundation.

REFERENCES Alfieri, A. A., Leite, J. P. G., Nakagomi, O., Kaga, E., Woods, P. A., Glass, R. I., and Gentsch, J. R. (1996). Characterization of human rotavirus genotype P[8]G5 from Brazil by probe-hybridization and sequence. Arch. Virol. 141, 2353–2364.

CHARACTERIZATION OF U.S. NONTYPEABLE ROTAVIRUS STRAINS Araujo, I. T., Ferreira, M. S. R., Failho, A. M., Assis, R. M., Cruz, C. M., Rocha, M., and Letie, J. P. G. (2001). Rotavirus genotypes P[4]G9, P[6]G9, and P[8]G9 in hospitalized children with acute gastorenteritis in Rio de Janeiro, Brazil. J. Clin. Microbiol. 39, 1999–2001. Arias, C. F., Ruiz, A. M., and Lopes, S. (1989). Further antigenic characterization of porcine rotavirus YM. J. Clin. Microbiol. 27, 2871–2873. Arista, S., Vizzi, E., Ferraro, D., Cascio, A., and Di Stefano, R. (1997). Distribution of VP7 serotypes and VP4 genotypes among rotavirus strains recovered from Italian children with diarrhea. Arch. Virol. 142, 2065–2071. Armah, G. E., Pager, C. T., Asmah, R. H., Anto, F. R., Oduro, A. R., Binka, F., and Steele, D. (2001). Prevalence of unusual human rotavirus strains in Ghanaian children. J. Med. Virol. 63, 67–71. Ball, J. M., Tian, P., Zeng, C. Q.-Y., Morris, A. P., and Estes, M. K. (1996). Age-dependent diarrhea induced by a rotaviral nonstructural glycoprotein. Science 272, 101–104. Bohl, E., Theil, K. W., and Saif, L. J. (1984). Isolation and serotyping of porcine rotaviruses and antigenic comparison with other rotaviruses. J. Clin. Microbiol. 19, 105–111. Bon, F., Fromantin, C., Aho, S., Pothier, P., and Kohli, E. (2000). G and P genotyping of rotavirus strains circulating in France over a three year period: Detection of G9 and P[6] strains at low frequencies. J. Clin. Microbiol. 38, 1681–1683. Browning, G. F., Chalmers, R. M., Fitzgerald, T. A., and Snodgrass, D. R. (1991a). Serological and genomic characterisation of L338, a novel equine group A rotavirus G serotype. J. Gen. Virol. 72, 1059–1064. Browning, G. F., Fitzgerald, T. A., Chalmers, R. M., and Snodgrass, D. R. (1991b). A novel group A rotavirus G serotype: Serological and genomic characterization of equine isolate F123. J. Clin. Microbiol. 29, 2043–2046. Browning, G. F., Snodgrass, D. R., Nakagomi, O., Kaga, E., Sarasini, A., and Gerna, G. (1992). Human and bovine serotype G8 rotaviruses may be derived by reassortment. Arch. Virol. 125, 121–128. Centers for Disease Control and Prevention (1999a). Withdrawal of rotavirus vaccine recommendation. MMWR 48(43), 1007. Centers for Disease Control and Prevention (1999b). Intussusception among recipients of rotavirus vaccine, United States, 1998–1999. MMWR 48(27), 577–581. Ciarlet, M., Liprandi, Conner, M. E., and Estes, M. K. (2000). Species specificity and interspecies relatedness of NSP4 genetic groups by comparative NSP4 sequence analyses of animal rotaviruses. Arch. Virol. 145, 371–383. Clark, H. F., Hoshino, Y., Bell, L. M., Groff, J., Hess, G., Bachman, P., and Offit, P. A. (1987). Rotavirus isolate W161 representing a presumptive new human serotype. J. Clin. Microbiol. 25, 1757–1762. Coulson, B., Unicomb, L. E., Pitson, G. A., and Bishop, R. F. (1987). Simple and specific enzyme immunoassay using monoclonal antibodies for serotyping human rotaviruses. J. Clin. Microbiol. 25, 509– 515. Coulson, B. S., Fowler, K. J., Bishop, R. F., and Cotton, R. G. H. (1985). Neutralizing monoclonal antibodies to human rotavirus and indications of antigenic drift among strains from neonates. J. Virol. 54, 14–20. Cubitt, W. D., Steele, A. D., and Iturriza, M. (2000). Characterisation of rotaviruses from children treated at a London hospital during 1996: Emergence of strains G9P2A[6] and G3P2A[6]. J. Med. Virol. 61, 150–154. Cunliffe, N. A., Woods, P. A., Ward, R. L., Leite, J. P. G., Das, B. K., Ramachandran, M., Bhan, M. K., Hart, C. A., Glass, R. I., and Gentsch, J. R. (1997). Sequence analysis of NSP4 gene of human rotavirus allows classification into two main genetic groups. J. Med. Virol. 53, 41–50. Cunliffe, N. A., Gondwe, J. S., Broadhead, R. L., Molyneux, M. E., Woods, P. A., Bresee, J. S., Glass, R. I., Gentsch, J. R., and Hart, C. A. (1999). Rotavirus G and P types in children with acute diarrhea in Blantyre, Malawi, from 1997 to 1998: Predominance of novel P[6]G8 strains. J. Med. Virol. 57, 308–312.

267

Cunliffe, N. A., Gentsch, J. R., Kirkwood, C. D., Dove, W., Nakagomi, O., Nakagomi, T., Hoshino, Y., Bresee, J. S., Glass, R. I., and Hart, C. A. (2000). Molecular and serologic characterization of novel serotype G8 rotavirus strains detected in Blantyre, Malawi. Virology 274, 309– 320. Das, B. K., Gentsch, J. R., Hoshino, Y., Ishida, S.-I., Nakagomi, O., Bhan, M. K., Kumar, R., and Glass, R. I. (1993). Characterization of the G serotype and genogroup of New Delhi newborn rotavirus strain 116E. Virology 197, 99–107. Das, B. K., Gentsch, J. R., Cicirello, H. G., Woods, P. A., Gupta, A., Ramachandran, M., Kumar, R., Bhan, M. K., and Glass, R. I. (1994). Characterization of rotavirus strains from newborns in New Delhi, India. J. Clin. Microbiol. 32, 1820–1822. Devereux, J., Haeberli, P., and Smithies, O. (1984). A comprehensive set of sequence analysis programs for the VAX. Nucleic Acids Res. 12, 387–395. Dunn, S. J., Greenberg, H. B., Ward, R. L., Nakagomi, O., Burns, J. W., Vo, P. T., Pax, K. A., Das, M., Gowda, K., and Rao, C. D. (1993). Serotypic and genotypic characterization of human serotype 10 rotaviruses from asymptomatic neonates. J. Clin. Microbiol. 31, 165–169. Estes, M. (1996). Rotaviruses and their replication. In “Fields Virology” (B. N. Fields, D. M. Knipe, and P. M. Howley, Eds.), 3rd ed., Vol. 2, pp. 1625–1655. Lippincott–Raven, Philadelphia. Estes, M. K. (2001). Rotaviruses and their replication. In “Fields Virology” (D. M. Knipe and P. M. Howley, Eds.), 4th ed., Vol. 2, pp. 1747–1786. Lippincott Williams & Wilkins, Philadelphia. Fischer, T. K., Steinsland, H., Molbak, K., Ca, R., Gentsch, J. R., Valentiner-Branth, P., Aaby, P., and Sommerfelt, H. (2000). Genotype profile of rotavirus strains from children in a suburban community in GuineaBissau, West Africa. J. Clin. Microbiol. 38, 264–267. Flores, J., Perez, I., White, L., Perez, M., Kalica, A. R., Marquina, R., Wyatt, R. G., Kapikian, A. Z., and Chanock, R. M. (1982). Genetic relatedness among human rotaviruses as determined by RNA hybridization. Infect. Immun. 37, 648–655. Garbarg-Chenon, A., Bricout, F., and Nicolas, J.-C. (1984). Study of genetic reassortment between two human rotaviruses. Virology 139, 358–365. Gentsch, J. R., Glass, R. I., Woods, P., Gouvea, V., Gorziglia, M., Flores, J., Das, B. K., and Bhan, M. K. (1992). Identification of group A rotavirus gene 4 types by polymerase chain reaction. J. Clin. Microbiol. 30, 1365–1373. Gentsch, J. R., Woods, P. A., Ramachandran, M., Das, B. K., Leite, J. P., Alfieri, A., Kumar, R., Bhan, M. K., and Glass, R. I. (1996). Review of G and P typing results from a global collection of strains: Implications for vaccine development. J. Infect. Dis. 174(Suppl. 1), S30–S36. Gerna, G., Sarasini, A., Di Matteo, A., Zentilin, L., Miranda, P., Parea, M., Baldanti, F., Arista, S., Milanesi, G., and Battaglia, M. (1990a). Serotype 3 human rotavirus strains with subgroup I specificity. J. Clin. Microbiol. 28, 1342–1347. Gerna, G., Sarasini, A., Zentilin, L., Di Matteo, A., Miranda, P., Parea, M., Battaglia, M., and Milanesi, G. (1990b). Isolation in Europe of 69Mlike (serotype 8) human rotavirus strains with either subgroup I or II specificity and a long RNA electropherotype. Arch. Virol. 112, 27–40. Gerna, G., Sarasini, A., Parea, M., Arista, S., Miranda, P., Brussow, H., Hoshino, Y., and Flores, J. (1992). Isolation and characterization of two distinct human rotavirus strains with G6 specificity. J. Clin. Microbiol. 30, 9–16. Gollop, R., Nakagomi, O., Silberstein, I., Shulman, L. M., Greenberg, H. B., Mendelson, E., and Shif, I. (1998). Three forms of AU-1 like human rotaviruses differentiated by their overall genomic constellation and by the sequence of their VP8. Arch. Virol. 143, 263–277. Gouvea, V., Glass, R. I., Woods, P., Taniguichi, K., Clark, H. F., Forrester, B., and Fang, Z. Y. (1990). Polymerase chain reaction amplification and typing of rotavirus nucleic acid from stool specimens. J. Clin. Microbiol. 28, 276–282. Green, K. Y., Hoshino, Y., and Ikegami, N. (1989). Sequence analysis of

268

GRIFFIN ET AL.

the gene encoding the serotype-specific glycoprotein (VP7) of two new human rotavirus serotypes. Virology 168, 429–433. Greenberg, H., McAuliffe, V., Valdesuso, J., Wyatt, R., Flores, J., Kalica, A., Hoshino, Y., and Singh, N. (1983). Serological analysis of the subgroup protein of rotavirus using monoclonal antibodies. Infect. Immun. 39, 91–99. Griffin, D. D., Kirkwood, C., Parashar, U. D., Woods, P. A., Bresee, J. S., Glass, R. I., Gentsch, J. R., et al. (2000). Surveillance of rotavirus strains in the United States: Identification of unusual strains. J. Clin. Microbiol. 38, 2784–2787. Holmes, J. L., Kirkwood, C. D., Gerna, G., Clemens, J. D., Rao, M. R., Naficy, A. B., Abu-Elyazeed, R., Savarino, S. J., Glass, R. I., and Gentsch, J. R. (1999). Characterization of unusual G8 rotavirus strains isolated from Egyptian children. Arch. Virol. 144, 1381–1396. Horie, Y., Nakagomi, O., Koshimura, Y., Nakagomi, T., Suzuki, Y., Oka, T., Sasaki, S., Matsuda, Y., and Watanabe, S. (1999). Diarrhea induction by rotavirus NSP4 in the homologous mouse model system. Virology 262, 398–407. Hoshino, Y., and Kapikian, A. Z. (1996). Classification of rotavirus VP4 and VP7 serotypes. Arch. Virol. 12(Suppl), 99–111. Hoshino, Y., Wyatt, R. G., and Greenberg, H. B. (1984). Serotypic similarity and diversity or rotaviruses of mammalian and avian origins as studies by plaque reduction neutralization. J. Infect. Dis. 149, 694– 702. Iizuka, M., Kaga, E., Chiba, M., Masamune, O., Gerna, G., and Nakagomi, O. (1994). Serotype G6 human rotavirus sharing a conserved genetic constellation with natural reassortants between members of the bovine and AU-1 genogroups. Arch. Virol. 135(3/4), 427–432. Isegawa, Y., Nakagomi, O., Nakagomi, T., and Ueda, S. (1992). A VP4 sequence highly conserved in human rotavirus strain AU-1 and feline rotavirus strain FRV-1. J. Gen. Virol. 73, 1939–1946. Kapikian, A. Z., and Chanock, R. M. (1990). Rotaviruses. In “Virology” (B. N. Fields, D. M. Knipe, R. M. Chanock, M. S. Hirsch, J. L. Melnick, T. P. Monath, and B. Roizman, Eds.), 2nd ed., Vol. 2, pp. 1353–1404. Raven Press, New York. Kapikian, A. Z., Flores, J., Vesikari, T., Ruuska, T., Madore, H. P., Green, K. Y., Gorziglia, M., Hoshino, Y., Chanock, R. M., Midthun, K., and Perez-Schael, I. (1991). Recent advances in development of a rotavirus vaccine for prevention of severe diarrheal illness of infants and young children. In “Immunology of Milk and the Neonate” (J. Mestecky, et al., Eds.), pp. 255–264. Plenum, New York. Kapikian, A. Z., Hoshino, Y., and Chanock, R. M. (2001). Rotaviruses. In “Fields Virology” (D. M. Knipe and P. M. Howley, Eds.), 4th ed., Vol. 2, pp. 1787–1833. Lippincott Williams and Wilkins, Philadelphia. Kelkar, S. D., and Ayachit, V. L. (2000). Circulation of group A rotavirus subgroups and serotypes in Pune, India 1990–1997. J. Health Popul. Nutr. 18, 163–170. Kirkwood, C. D., and Palombo, E. A. (1997). Genetic characterization of the rotavirus nonstructural protein, NSP4. Virology 236, 258–265. Kirkwood, C. D., Gentsch, J. R., Hoshino, Y., Clark, H. F., and Glass, R. I. (1999). Genetic and antigenic characterization of a serotype P[6]G9 human rotavirus strain isolated in the U.S. Virology 256, 45–53. Leite, J. P., Alfieri, A. A., Woods, P., Glass, R. I., and Gentsch, J. R. (1996). Rotavirus G and P types circulating in Brazil: Characterization by RT-PCR, probe hybridization, and sequence analysis. Arch. Virol. 141, 2365–2374. Maneekarn, N., and Ushijima, H. (2000). Epidemiology of rotavirus infections in Thailand. Pediatr. Int. 42, 415–421. Matsuno, S., Hasegawa, A., Mukoyama, A., and Inouye, S. (1985). A candidate for a new serotype of human rotavirus. J. Virol. 54, 623– 624. McNulty, M. S., Allan, G. M., Todd, D., and McFerran, J. B. (1979). Isolation and cell culture propagation of neonatal calf diarrhea (scours) virus. Arch. Virol. 61, 13–21. Mebus, C. A., Kono, M., Underdahl, N. R., and Twiehaus, M. J. (1971). Cell culture propagation of neonatal calf diarrhea (scours) virus. Can. Vet. J. 12, 69–72.

Merril, C. R., Goldman, D., Sedman, S. A., and Ebert, M. H. (1981). Ultrasensitive stain for proteins in polyacrylamide gels shows regional variation in cerebrospinal fluid proteins. Science 211, 1437. Murphy, T. V., Gargiullo, P. M., Massoudi, M. S., Nelson, D. B., Jumaan, A. O., Okoro, C. A., Zanardi, L. R., Setia, S., Fair, E. L., LeBaron, C. W., Schwartz, B., Wharton, M., and Livingood, J. R. (2001). Intussusception among infants given an oral rotavirus vaccine. N. Engl. J. Med. 344, 564–572. Nakagomi, O., and Nakagomi, T. (1991). Molecular evidence for naturally occurring single VP7 gene substitution reassortant between human rotaviruses belonging to two different genogroups. Arch. Virol. 119, 67–81. Nakagomi, O., and Nakagomi, T. (1993). Interspecies transmission of rotaviruses studied from the perspective of genogroup. Microbiol. Immunol. 37, 337–348. Nakagomi, O., Nakagomi, T., Hoshino, Y., Flores, J., and Kapikian, A. Z. (1987). Genetic analysis of a human rotavirus that belongs to subgroup 1 but has an RNA pattern typical of subgroup II human rotaviruses. J. Clin. Microbiol. 25, 1159–1164. Nakagomi, O., Nakagomi, T., Akatani, K., and Ikegami, N. (1989). Identification of rotavirus genogroups by RNA–RNA hybridization. Mol. Cell. Probes 3, 251–261. Nakagomi, O., Kaga, E., Gerna, G., Sarasini, A., and Nakagomi, T. (1992a). Subgroup I serotype 3 human rotavirus strains with long RNA pattern as a result of naturally occurring reassortment between members of the bovine and AU-1 genogroups. Arch. Virol. 126, 337–342. Nakagomi, O., Kaga, E., and Nakagomi, T. (1992b). Human rotavirus strain with unique VP4 neutralization epitopes as a result of natural reassortment between members of the AU-1 and Wa genogroups. Arch. Virol. 127, 365–371. Nakagomi, O., Isegawa, Y., Ueda, S., Gerna, G., Sarasini, A., Kaga, E., Nakagomi, T., and Flores, J. (1993). Nucleotide sequence comparison of the VP8 gene of rotaviruses possessing the AU-1 gene 4 allele. J. Gen. Virol. 74, 1709–1713. Nakagomi, T. (2000). Rotavirus infection and intussusception: A view from retrospect. Microbiol. Immunol. 44, 619–628. O’Halloran, F., Lynch, M., Cryan, B., O’Shea, H., and Fanning, S. (2000). Molecular characterization of rotavirus in Ireland: Detection of novel strains circulating in the population. J. Clin. Microbiol. 38, 3370–3374. Oka, T., Nakagomi, T., and Nakagomi, O. (2000). Apparent re-emergence of serotype G9 in 1995 among rotaviruses recovered from Japanese children hospitalized with acute gastroenteritis. Microbiol. Immunol. 44, 957–961. Pager, C. T., Alexander, J. J., and Steele, A. D. (2000). South African G4P(6) asymptomatic and symptomatic neonatal rotavirus strains differ in their NSP4, VP8*, and VP7 genes. J. Med. Virol. 62, 208–216. Palombo, E. A., and Bishop, R. F. (1995). Genetic and antigenic characterization of a serotype G6 human rotavirus isolated in Melbourne, Australia. J. Med. Virol. 47, 348–354. Palombo, E. A., Bugg, H. C., Masendycz, P. J., Coulson, B. S., Barnes, G. L., and Bishop, R. F. (1996). Multiple-gene rotavirus reassortants responsible for an outbreak of gastroenteritis in central and northern Australia. J. Gen. Virol. 77, 1223–1227. Palombo, E. A., Masendycz, P. J., Bugg, H. C., Bogdanovic-Sakran, N., Barnes, G. L., and Bishop, R. F. (2000). Emergence of serotype G9 human rotaviruses in Australia. J. Clin. Microbiol. 38, 1305–1306. Parashar, U. D., Holman, R. C., Clarke, M. J., Bresee, J. S., and Glass, R. I. (1997). Hospitalizations associated with rotavirus diarrhea in the United States, 1993 through 1995: Surveillance based on the new ICD-9-CM rotavirus-specific diagnostic code. J. Infect. Dis. 177, 13– 17. Piec, T. L., and Palombo, E. A. (1997). Sequence comparison of the VP7 of serotype G2 rotaviruses from diverse geographical locations. DNA Sequence 8, 1–5. Ramachandran, M., Das, B. K., Vij, A., Kumar, R., Bhambal, S. S., Kesari, N., Rawat, H., Bahl, L., Thakur, S., Woods, P. A., Glass, R. I., Bhan,

CHARACTERIZATION OF U.S. NONTYPEABLE ROTAVIRUS STRAINS M. K., and Gentsch, J. R. (1996). Unusual diversity of human rotavirus G and P genotypes in India. J. Clin. Microbiol. 34, 436–439. Ramachandran, M., Gentsch, J. R., Parashar, U. D., Jin, S., Woods, P. A., Holmes, J. L., Kirkwood, C. D., Bishop, R. F., Greenberg, H. B., Urasawa, S., Gerna, G., Coulson, B. S., Taniguchi, K., Bresee, J. S., Glass, R. I., and The National Rotavirus Strain Surveillance System Collaborating Laboratories (1998). Detection and characterization of novel rotavirus strains in the United States. J. Clin. Microbiol. 36, 3223–3229. Ramachandran, M., Kirkwood, C. D., Unicomb, L., Cunliffe, N. A., Ward, R. L., Bhan, M. K., Clark, H. F., Glass, R. I., and Gentsch, J. R. (2000). Molecular characterization of serotype G9 rotavirus strains from a global collection. Virology 278, 436–444. Rasool, N. B. G., Larralde, G., and Gorziglia, M. I. (1993). Determination of human rotavirus VP4 using serotype-specific cDNA probes. Arch. Virol. 133, 275–282. Santos, N., Lima, R. C. C., Pereira, C. F. A., and Gouvea, V. (1998). Detection of rotavirus types G8 and G10 among Brazilian children with diarrhea. J. Clin. Microbiol. 36, 2727–2729. Santos, N., Volotao, E. M., Soares, C. C., Albuguerque, M. C. M., Da Silva, F. M., De Carvalho, T. R. B., Pereira, C. F. A., Chizhikov, V., and Hoshino, Y. (2001). Rotavirus strains bearing genotype G9 or P[9] recovered from Brazilian children with diarrhea from 1997 to 1999. J. Clin. Microbiol. 39, 1157–1160. Shif, I., Iizuka, M., Silberstein, I., Mendelson, E., and Nakagomi, O. (1994). Rotaviruses belonging to the AU-1 genogroup recovered from Israeli infants with diarrhea. Arch. Virol. 138, 157–164. Snodgrass, D. R., Fitzgerald, T., Campbell, I., Scott, F. M. M., Browning, G. F., Miller, D. L., Herring, A. J., and Greenberg, H. B. (1990). Rotavirus serotypes 6 and 10 predominate in cattle. J. Clin. Microbiol. 28, 504–507. Steele, A. D., Garcia, D., Sears, J., Gerna, G., Nakagomi, O., and Flores, J. (1993). Distribution of VP4 gene alleles in human rotaviruses by using probes to the hyperdivergent region of the VP4 gene. J. Clin. Microbiol. 31, 1735–1740. Stuker, G., Oshiro, L. S., and Schmidt, N. J. (1980). Antigenic comparisons of two new rotaviruses from rhesus monkeys. J. Clin. Microbiol. 11, 202–203. Taniguchi, K., Urasawa, T., Morita, Y., Greenberg, H. B., and Urasawa, S. (1987). Direct serotyping of human rotavirus in stools using serotype 1-, 2-, 3-, and 4-specific monoclonal antibodies to VP7. J. Infect. Dis. 155, 1159–1166. Taniguchi, K., Urasawa, T., Kobayshi, N., Gorziglia, M., and Urasawa, S. (1990). Nucleotide sequence of VP4 and VP7 genes of human rotaviruses with subgroup I specificity and long RNA pattern: Implication for new G serotype specificity. J. Virol. 64, 5640–5644. Tucker, A. W., Haddix, A. C., Bresee, J. S., Holman, R. C., Parashar, U. D., and Glass, R. I. (1998). Cost-effectiveness analysis of a rotavirus immunization program for the United States. J. Am. Med. Assoc. 279, 1371–1376. Unicomb, L. E., Podder, G., Gentsch, J. R., Woods, P. A., Hasan, K. Z., Faruque, A. S. G., Albert, M. J., and Glass, R. I. (1999). Evidence of

269

high-frequency genomic reassortment of group A rotavirus strains in Bangladesh: Emergence of type G9 in 1995. J. Clin. Microbiol. 37, 1885–1891. Urasawa, S., Urasawa, T., Wakasugi, F., Kobayashi, N., Taniguchi, K., Lintag, I. C., Saniel, M. C., and Goto, H. (1990). Presumptive seventh serotype of human rotavirus. Arch. Virol. 113, 279–282. Urasawa, T., Taniguchi, K., Kobayashi, N., Mise, K., Hasegawa, A., Yamazi, Y., and Urasawa, S. (1993). Nucleotide sequence of VP4 and VP7 genes of a unique human rotavirus strain Mc35 with subgroup I and serotype 10 specificity. Virology 195, 766–771. Van Man, N., Van Trang, N., Lien, H. P., Trach, D. D., Thanh, N. T. H., Tu, P., Long, N. T., Luan, L. T., Ivanoff, B., Gentsch, J. R., Glass, R. I., and Vietnam Rotavirus Surveillance Network (2001). The epidemiology and disease burden of rotavirus in Vietnam: Sentinel surveillance at 6 hospitals. J. Infect. Dis. 183, 1707–1712. Ward, R. L., Nakagomi, O., Knowlton, D. R., McNeal, M. M., Nakagomi, T., Clemens, J. D., Sack, D. A., and Schiff, G. M. (1990). Evidence for natural reassortants of human rotaviruses belonging to different genogroups. J. Virol. 64, 3219–3225. Wen, L., Ushijima, H., Kakizawa, J., Fang, Z.-Y., Nishio, O., Morikawa, S., and Motohiro, T. (1995). Genetic variation in VP7 gene of human rotavirus serotype (G2 type) isolated in Japan, China, and Pakistan. Microbiol. Immunol. 39, 911–915. Widdowson, M.-A., Van Doornum, G. J. J., Van der Poel, W. H. M., De Boer, A. S., Mahdi, U., and Koopmans, M. (2001). Emerging group-A rotavirus and a nosocomial outbreak of diarrhoea. Lancet 356, 1161– 1162. Woode, G. N., Jones, J., and Brdiger, J. (1975). Levels of colostral antibodies against neonatal calf diarrhea virus. Vet. Rec. 97, 148–149. Woode, G. N., Kelso, N. E., Simpson, T. F., Gaul, S. K., Evans, L. E., and Babiuk, L. (1983). Antigenic relationships among some bovine rotaviruses: Serum neutralization and cross-protection in gnotobiotic calves. J. Clin. Microbiol. 18, 358–364. Woods, P. A., Gentsch, J., Gouvea, V., Mata, L., Simhon, A., Santosham, M., Bai, Z.-S., Urasawa, S., and Glass, R. I. (1992). Distribution of serotypes of human rotavirus in different populations. J. Clin. Microbiol. 30, 781–785. Wyatt, R. G., James, H. D., Jr., Pittmann, A. L., Hoshino, Y., Greenberg, H. B., Kalica, A. R., Flores, J., and Kapikian, A. Z. (1983). Direct isolation in cell culture of human rotaviruses and their characterization into four serotypes. J. Clin. Microbiol. 18, 310–317. Wyatt, R. G., Greenberg, H. B., and James, W. D. (1984). Definition of human rotavirus serotypes by plaque reduction assay. Infect. Immun. 37, 110–115. Zhang, M., Zeng, C. Q., Dong, Y., Ball, J. M., Saif, L. J., Morris, A. P., and Estes, M. K. (1998). Mutations in rotavirus nonstructural glycoprotein NSP4 are associated with altered virus virulence. J. Virol. 72, 3666– 3672. Zhou, Y., Li, L., Kim, B., Kaneshi, K., Nishimura, S., Kuroiwa, T., Nishimura, T., Sugita, K., Ueda, Y., Nakaya, S., and Ushijima, H. (2000). Rotavirus infection in children in Japan. Pediatr. Int. 42, 428–439.