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Journal of Virology, April 2008, p. 4175-4179, Vol. 82, No. 8
0022-538X/08/$08.00+0     doi:10.1128/JVI.02537-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

The VP7 Genes of Two G9 Rotaviruses Isolated in 1980 from Diarrheal Stool Samples Collected in Washington, DC, Are Unique Molecularly and Serotypically ^,

Dianjun Cao,¹ Norma Santos,^1,2 Ronald W. Jones,¹ Masatoshi Tatsumi,^1, Jon R. Gentsch,³ and Yasutaka Hoshino^1*

Epidemiology Section, Laboratory of Infectious Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland 20892,¹ Departamento de Virologia, Instituto de Microbiologia, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil,² Gastroenteritis and Respiratory Viruses Laboratory Branch, Centers for Disease Control and Prevention, Atlanta, Georgia 30333³

Received 27 November 2007/ Accepted 19 January 2008

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In a retrospective study of archival diarrheal stool samples collected from 1974 to 1991 at Children's Hospital National Medical Center, Washington, DC, we detected three genotype G9P[8] viruses in specimens collected in 1980, which represented the earliest human G9 viruses ever isolated. The VP7 genes of two culture-adapted 1980 G9 viruses were phylogenetically related closely to the lineage 2 G9 virus VP7 gene. Unexpectedly, however, the VP7s of the 1980 G9 viruses were more closely related serotypically to lineage 3 VP7s than to lineage 2 VP7, which may be supported by amino acid sequence analyses of the VP7 proteins.

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Rotaviruses are the single most important etiologic agents of severe infantile diarrhea worldwide, and the development and implementation of a safe and effective vaccine have been important global public health goals (1, 8, 13, 18). Although 11 G (VP7) types and 12 P (VP4) types have been detected in human rotaviruses, G1 to G4 and G9 and P[4], P[6], and P[8] are the most commonly detected human rotavirus specificities worldwide(for reviews, see references 9, 15, and 22). Serotype G9 rotaviruses continue to attract special attention because the G9 viruses have a unique natural history and evolution in human communities (for reviews, see references 10 and 11). The G9 rotavirus VP7 gene belongs to one of at least three phylogenetic lineages (16, 17, 21, 23, 24): lineage 1 (strains isolated in the 1980s in the United States and Japan), lineage 2 (strains first isolated in 1986 and exclusively in India thus far), and lineage 3 (strains that emerged/reemerged in the mid-1990s). Recently, Phan et al. (20) proposed to establish three additional lineages, lineages 4, 5, and 6. However, we found, judging from our phylogenetic as well as codon usage analyses, that lineages 4, 5, and 6 could be regarded as sublineages of lineages 1, 2, and 3, respectively; thus, in this paper we follow the traditional classification system of lineages 1 to 3.

During a study of rotavirus strains found in archival diarrheal stool samples collected longitudinally (1974 to 1991) from hospitalized children at Children's Hospital National Medical Center in Washington, DC, we detected three G9P[8] viruses from specimens collected in 1980 (2; N. Santos et al., unpublished data). This was an exciting finding, since the earliest available G9 rotavirus was isolated in 1983 from a stool sample collected from a child with diarrhea in Philadelphia, PA (6). Two (strains DC706 and G2275) of the 1980 G9 viruses were successfully adapted to growth in cell culture, which represented the earliest human G9 viruses ever isolated.

Sequence and phylogenetic analyses. Full-length cDNAs of the VP7 genes of the culture-adapted strains DC706 and G2275 were amplified and sequenced as described previously (10). The VP7 genes of the DC706 and G2275 and other selected G9 rotavirus strains were analyzed using the MEGA 4.0 software. Sequence alignment was performed using Clustal W, genetic distance was calculated using the Kimura two-parameter method, and the phylogenetic tree was constructed using the neighbor-joining method with 1,000 bootstrap repetitions. We calculated the relative synonymous codon usage value of each of the 27 G9 virus VP7 genes and analyzed such relative synonymous codon usage values by the average linkage cluster method of hierarchical cluster analysis. The 27 G9 strains analyzed are as follows (strain name followed by GenBank accession number in parentheses): DC706 (EU153553), G2275 (EU153554), WI61 (AB180969), AU32 (AB045372), F45 (AB180970), 116E (L14072), Om46 (AJ491181), Om67 (AJ491179), INL1 (AJ250277), G16 (AJ250276), RMC321 (AF501578), Mc345 (D38055), US1205 (AF060487), B5544 (AY487871), B4589 (AY487889), R143 (AF274969), R44 (AF438227), BD524 (AJ250543), BD431 (AJ250542), Bulumkutu (AF359358), CIT-254 (AF281044), T203 (AY003871), JP35-7 (AB176683), JP3-6 (AB176678), SP1904 (AB091754), A2 (AB180978), and 97'SZ37 (AF260959).

The 1980 G9 strains DC706 and G2275 shared 99.9% VP7 gene nucleotide identity and together shared the highest identity (92.5 to 92.6%) with strain 116E (lineage 2) and strain Om67, which was isolated in 1997 to 1998 from a child with diarrhea in Omaha, NE (15), followed by lineage 1 viruses (91.1 to 92.0%) and lineage 3 viruses (88.6 to 91.0%) (Table 1). The two 1980 G9 strains shared VP7 amino acid identity of 99.4% and together had the highest amino acid identity with strain Om67 (96.6 to 96.9%), followed by lineage 1 viruses (95.1 to 96.9%), lineages 3 viruses (92.4 to 95.4%), and lineage 2 strain 116E (91.7 to 92.0%).

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TABLE 1. Nucleotide and deduced amino acid identities among the VP7s of G9 strains isolated in 1980 and 1 porcine and 11 human G9 rotavirus strains belonging to VP7 phylogenetic lineage 1, 2, or 3

Phylogenetic analyses of the VP7 gene nucleotide sequences showed that the DC706 and G2275 strains grouped together with strain 116E to form lineage 2 and that the three strains together displayed a close relationship to the Om67 strain (Fig. 1). Lineage 3 virus VP7 genes were confirmed to be distinct phylogenetically from those of lineage 1 and 2 viruses. Among lineage 3 viruses, human virus VP7 genes tended to form one cluster whereas porcine virus VP7 genes formed another cluster, except for VP7 genes of human virus strains T203 and SP1904, which grouped together with porcine virus VP7 genes. Moreover, codon usage bias analysis confirmed the genetic and evolutionary relationships among G9 virus VP7 genes predicted by phylogenetic tree analyses (see Table S1 and Fig. S1 in the supplemental material).

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FIG. 1. Phylogenetic tree for VP7 genes of selected serotype G9 rotavirus strains rooted to that of the KUN (G2) strain.

Neutralization characterization of the VP7 proteins of the 1980 G9 viruses and Om67 virus. Since the interaction of the VP4 to VP7 outer capsid proteins of rotavirus has been reported to affect the expression of selected phenotypes of one or both proteins (3, 4, 19), we constructed three single VP7 gene substitution rotavirus reassortants, each of which had 10 genes of bovine rotavirus strain UK and only the VP7 gene of human G9 rotavirus strain DC706, G2275, or Om67. The generation and characterization of reassortants UK x WI61, UK x AU32, UK x 116E, UK x R44, UK x R143, UK x INL1, and UK x BD524 were reported previously (11). Table 2 summarizes the antigenic characterization of outer capsid glycoprotein VP7 of the 1980 G9 strains DC706 and G2275 as well as the Om67 strain in relation to selected G9 strains belonging to VP7 gene phylogenetic sequence lineage 1, 2, or 3 by analysis of their guinea pig hyperimmune antiserum neutralization profiles. Antiserum to the VP7 protein of strain DC706, G2275, or Om67 neutralized lineage 3 viruses within twofold of the homotypic viruses and neutralized lineage 1 and 2 viruses 32- to 1,024-fold less efficiently than the homotypic viruses, indicating that the VP7 proteins of these three G9 strains analyzed were closely related antigenically to lineage 3 viruses and distantly related to lineage 1 and 2 viruses. The extent of the antigenic relatedness among these viruses was analyzed further by examining the reciprocal neutralization specificities: (i) antisera to the lineage 3 virus VP7s neutralized the DC706, G2275, and Om67 viruses to a high titer, indicating that these three viruses and lineage 3 viruses were similar, if not identical, in both directions, as determined by the 20-antibody criterion for relatedness (7, 12, 14), and (ii) antisera to lineage 1 (WI61 and AU32) and 2 (116E) virus VP7s neutralized the DC706, G2275, and Om67 strains as efficiently as the homotypic virus strains, indicating that the WI61, AU32, and 116E strains were the prime strains for these three strains (i.e. 20 U of antibody to the WI61, AU32, and 116E strains would recognize the DC706, G2275, and Om67 strains but not vice versa). This was unexpected, since as stated above, both phylogenetic and codon usage bias analyses indicated that the VP7 genes of the DC706, G2275, and Om67 strains were most distantly related to those of lineage 3 viruses. In addition, the 1980 G9 viruses were found to share the lowest VP7 gene nucleotide identity with lineage 3 viruses (88.6 to 89.9%). However, at the amino acid level, although the overall VP7 amino acid identity of the 1980 G9 viruses and Om67 virus was higher versus lineage 1 viruses (95.1 to 96.9%) than versus lineage 3 viruses (92.4 to 96.3%), an amino acid sequence alignment (Fig. 2) suggested that the VP7 proteins of the 1980 G9 and Om67 viruses may be more similar antigenically to those of lineage 3 viruses than to those of lineage 1 viruses. For example, mutations detected in antigenic regions A (VR5) and F (VR9) were shared only among the 1980 G9 strains, strain Om67, and lineage 3 strains, which may explain the close neutralization relationships detected among them in this study. Single point mutations in antigenic regions A and/or F selected with G11-specific monoclonal antibodies have been reported to affect the reactivity of the variants with polyclonal antibodies (5). Finally, from the point of view of the development of G9 rotavirus vaccine candidates, the lineage 1 VP7s were confirmed in this study to be the best in inducing the most broadly reactive neutralizing antibodies (11).

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TABLE 2. Antigenic characterization of outer capsid glycoprotein VP7s of rotavirus strains DC706, G2275, and Om67 in relation to selected G9 strains belonging to VP7 gene phylogenetic sequence lineage 1, 2, or 3

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FIG. 2. Comparison of the deduced amino acid sequences of the VP7s of the 1980 G9 strains and 12 serotype G9 strains employed in the present study. Three VRs (VR1 to VR3) which are not antigenic sites are shaded. Six VRs (VR4 to VR9) and eight amino acid residues which have been demonstrated to be involved in the formation of antigenic sites (shown as letters in parentheses) through epitope mapping studies (reviewed in reference 13) are not shaded.

In summary, by characterizing phylogenetically the VP7 genes of a total of 27 G9 rotavirus strains, we showed that the VP7 genes of lineage 2 strains DC706, G2275, and 116E may represent a progenitor VP7 gene for lineage 2 Om67 and lineage 1 virus VP7 genes. In addition, we demonstrated that the VP7 proteins of the 1980 G9 viruses were similar, if not identical, serotypically to those of phylogenetically distantly related lineage 3 G9 viruses, indicating that a phenotypic (i.e., neutralization) relatedness of rotavirus VP7 proteins was independent of a phylogenetic relatedness of VP7 genes encoding such proteins.

 
We thank Jerri Ross for expert technical assistance; Albert Z. Kapikian for the continuing support of our work; and H. Fred Clark, Osamu Nakagomi, and Nobuko Ikegami for providing us with rotavirus strains WI61, AU32, and F45, respectively.

This work was supported in part by the Intramural Research Program of National Institute of Allergy and Infectious Disease, National Institutes of Health.

There is no conflict of interest to declare.

The findings and conclusions in this report are those of the authors and do not necessarily represent the views of CDC.

 
* Corresponding author. Mailing address: Epidemiology Section, Laboratory of Infectious Diseases, NIAID, NIH, Building 50, Room 6308, Bethesda, MD 20902. Phone: (301) 594-1851. Fax: (301) 480-1387. E-mail: thoshino{at}niaid.nih.gov

Published ahead of print on 30 January 2008.

Supplemental material for this article may be found at http://jvi.asm.org/.

Present address: Department of Pediatrics, Sapporo Medical University, Sapporo, Japan.

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Journal of Virology, April 2008, p. 4175-4179, Vol. 82, No. 8
0022-538X/08/$08.00+0     doi:10.1128/JVI.02537-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

This Article Abstract Full Text (PDF) Supplemental material Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Reprints and Permissions Copyright Information Books from ASM Press MicrobeWorld Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Cao, D. Articles by Hoshino, Y. Search for Related Content PubMed PubMed Citation Articles by Cao, D. Articles by Hoshino, Y.