This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowReprints and Permissions
Right arrow Copyright Information
Right arrow Books from ASM Press
Right arrow MicrobeWorld
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Araújo, I. T.
Right arrow Articles by Leite, J. P. G.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Araújo, I. T.
Right arrow Articles by Leite, J. P. G.

 Previous Article  |  Next Article 

Journal of Clinical Microbiology, May 2001, p. 1999-2001, Vol. 39, No. 5
0095-1137/01/$04.00+0   DOI: 10.1128/JCM.39.5.1999-2001.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.

Rotavirus Genotypes P[4]G9, P[6]G9, and P[8]G9 in Hospitalized Children with Acute Gastroenteritis in Rio de Janeiro, Brazil

I. T. Araújo,1 M. S. R. Ferreira,1 A. M. Fialho,1 R. M. Assis,1 C. M. Cruz,1 M. Rocha,2 and J. P. G. Leite1,*

Departamento de Virologia, Instituto Oswaldo Cruz,1 and Hospital Municipal Jesus,2 Rio de Janeiro, Rio de Janeiro, Brazil

Received 5 October 2000/Returned for modification 2 January 2001/Accepted 5 March 2001


    ABSTRACT
Top
Abstract
Text
References

Fifty-three rotavirus-positive fecal specimens from children with diarrhea admitted to a Rio de Janeiro, Brazil, children's hospital between January 1997 and December 1998 were characterized for P and G types by using reverse transcription-PCR. Genotype P[4]G2 accounted for 21% of isolates, while uncommon genotypes P[8]G9, P[6]G9, and P[4]G9 accounted for 13% of the isolates.


    TEXT
Top
Abstract
Text
References

Group A rotaviruses (RV) are the leading cause of severe gastroenteritis in infants and young children worldwide and are associated with 600,000 to 800,000 deaths each year, mostly in developing countries (9).

Belonging to the Reoviridae family, RV are characterized by the presence of 11 segments of double-stranded RNA (dsRNA) enclosed in a triple-shelled protein capsid. Serotypes of RV are defined by the two outer proteins: the protease-sensitive protein, VP4, and the major glycoprotein, VP7. The two outer proteins induce neutralizing antibodies and designate the G (VP7) and P (VP4) viral serotypes (8). Using genotyping methods to determine P and G genotypes, the most common combinations found in humans worldwide are P[8]G1, P[4]G2, P[8]G3, and P[8]G4 (9). Each serotype of RV induces serotype-specific protective immunity, which has been used as the basis for the development of an RV vaccine including the four most important human strains (13). However, uncommon genotypes and/or serotypes infecting humans in developed and developing countries throughout the world have been detected (9, 19). In Brazil, uncommon RV serotypes and/or genotypes, such as P[8]G5, P[6]G2, G8, G10, mixed infections, and a significant number of untypeable strains, have been previously reported (14, 20). Serotype G9 RV was first described in the United States (4) and later in different localities, including Japan (15), Thailand (22), India (18), Italy (1), Bangladesh (21), and Malawi (6). Griffin et al. (12) detected and characterized G9 RV strains in 10 of 12 cities in the United States and suggested that this strain could be emerging as significant, reaching epidemic proportions. Other recent studies support the increasing importance of serotype G9 as a cause of severe diarrhea in children around the world (2, 5, 16, 17). Therefore, serotype G9 must be taken into consideration for prospective RV vaccine studies, since this serotype is not constituted in any of the preceding RV vaccines.

Considering the large diversity of RV infecting Brazilian children, we report strain genotyping results from 53 positive RV fecal specimens collected from hospitalized children less than 3 years old with acute gastroenteritis at Hospital Municipal Jesus (Rio de Janeiro, RJ, Brazil), a reference center for children care. The samples were collected between January 1997 and December 1998 during the first 8 h of hospitalization, and the children took medical care for at least 2 days. Approximately 10% (wt/vol) stool suspensions were prepared in Tris-HCl Ca2+ (0.01 M) (pH 7.2), and an enzyme immunoassay for RV and adenovirus antigen detection (EIARA) was carried out. Concomitantly, stool suspensions were used for dsRNA extraction by the glass powder method (3), followed by a polyacrylamide gel electrophoresis (PAGE). Electrophoresed dsRNA segments were visualized by silver staining. The EIARA, PAGE, and silver staining procedures were performed as previously described (14).

The viral dsRNA, extracted from RV-positive clarified stool supernatants by the glass powder method (3), was first reverse transcribed (RT) and amplified by PCR (first amplification step) with a pair of consensus primers corresponding to a conserved nucleotide sequence of the VP7 (7, 11) or VP4 (10) genes. The DNA fragment obtained of 904 bp (VP7) or 876 bp (VP4) was then used as a template in a second PCR, carried out by using 1 µl of the first amplicon and a pool of genotype-specific primers complementary to variable regions of the VP7 (11) or VP4 (10) gene. Temperature and time conditions for PCR amplifications were performed as originally described (10, 11). Distilled Milli-Q water was used as a negative control in all techniques, and recommended manipulations for PCR procedures were carried out as a precaution to avoid false-positive results.

The P[4]G2 genotype was the most common strain detected in 21% of these isolates, followed by P[8]G2 (17%), P[8]G1 (11%), P[8]G3 (6%), P[8]G4 (6%), P[8]G10 (6%), P[6]G4 (4%), and one mixed infection by P[8+4]G3 (2%). In addition, we detected seven G9 strains: five P[8]G9 (9%), one P[6]G9 (2%), and one P[4]G9 (2%). As shown in Fig. 1, DNA bands corresponding to amplified segments of 110 bp for genotype G9 were visualized. The G9 strains were confirmed using Southern hybridization and chemiluminescent detection (data not shown), performed as previously described (14). The 5'-end-labeled digoxigenin-oligonucleotide probe for the G9 gene strain US1205 (5' GC ATC AAC TCA AAT TGG AGA T; VP7 gene, nucleotides 313 to 333, plus-sense) was designed to be homologous to internal regions of the consensus G9 first-round RT-PCR product produced by primers 9con1 and 9con2. In addition, we sequenced the G9 genotype-specific PCR products with the Big Dye Terminator (PE Biosystem Inc.) on an automated sequencer, ABI 377. The sequenced Brazilian strains were very similar to United States and Malawi G9 strains (data not shown), suggesting that they belong to type G9. The nucleotide and amino acid homology between Brazilian strain 1527 and other G serotypes showed very high homology to prototype G9 strain US1205 (Table 1), confirming that these strains belong to serotype G9.


View larger version (42K):
[in this window]
[in a new window]
  		FIG. 1.   Nested PCR products for seven samples characterized as G9 genotype. Lane M, 123-bp ladder molecular size marker (Life Technologies, Inc.); lane XIII, molecular weight marker type XIII, digoxigenin labeled (Boehringer Mannheim Biochemicals); lanes 1 through 7, Brazilian strains of genotype G9; lane NC, negative control.

                              
View this table:
[in this window]
[in a new window]
  		TABLE 1.   Nucleotide and amino acid homology between Brazilian strain 1527 (nucleotides 28 to 1034, amino acids 1 to 326) and the VP7 gene of other rotavirus G serotypes

Analysis of dsRNA by PAGE and silver staining revealed that all P[8] and P[4] strains had long electropherotypes, while the P[6] strain had a short profile. Samples that could not be characterized for G type (4 samples), P type (1 sample), or G and P types (3 samples) represented 14% of the strains. Despite the methodologies used in this study, some strains remain untypeable, raising the possibility that additional genotypes may be present in the population. It is important to characterize these strains, since they may emerge as being predominant in the future, as type G9 did between March and May of 1998 in the present work. The present study reports for the first time G9 strains infecting humans in Brazil; thus, these findings have significant implications, since this serotype has recently emerged in Australia (17), France (2), England (5), Ireland (16), and the United States (12), raising the possibility that RV type G9 may represent a fifth globally important serotype. The identification of novel P and G combinations of RV in Rio de Janeiro emphasizes the ability of this segmented virus to form reassortants (21) which may result in the establishment of new important strains.

Nucleotide sequence accession number. The VP7 gene sequence of Brazilian RV strain 1527 has been assigned the accession number AJ279082.


    ACKNOWLEDGMENTS

This work was supported by FIOCRUZ, COLAB/MS, and CNPq.

We thank members of the WHO/PAHO Rotavirus Collaborating Center in the Viral Gastroenteritis Section of the Centers for Disease Control and Prevention (Dixie Griffin and Jon Gentsch) for assistance with this investigation and for critical review of the manuscript (Jon Gentsch).


    ADDENDUM IN PROOF

Since the submission of this report, N. Santos et al. (J. Clin. Microbiol. 39:1157-1160, 2001) reported rotavirus serotype G9 in the state of Rio de Janeiro from 1997 to 1999, and the present study reinforces the previous description with the identification of G9 strains infecting hospitalized children (inpatient).


    FOOTNOTES

* Corresponding author. Mailing address: Laboratório de Virologia Comparada, Departamento de Virologia, FIOCRUZ, Av. Brazil, 4365, 21045-900, Rio de Janeiro, RJ, Brazil. Phone: 55-21-5984417. Fax: 55-21-5984491. E-mail: jpgleite{at}ioc.fiocruz.br.


    REFERENCES
Top
Abstract
Text
References

1. Arista, S., E. Vizzi, D. Ferraro, A. Cascio, and R. Di Stefano. 1997. Distribution of VP7 serotypes and VP4 genotypes among rotavirus strains recovered from Italian children with diarrhea. Arch. Virol. 142:2065-2071[CrossRef][Medline].
2. Bon, F., S. Fromantin, S. Aho, P. Pothier, E. Kohli, and The Azay Group. 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[Abstract/Free Full Text].
3. Boom, R., C. J. A. Sol, M. M. M. Salimans, C. L. Jansen, P. M. E. Wertheim-van Dillen, and J. Van der Noordaa. 1990. Rapid and simple method for purification of nucleic acids. J. Clin. Microbiol. 28:495-503[Abstract/Free Full Text].
4. Clark, H. F., Y. Hoshino, L. M. Bell, J. Groff, G. Hess, P. Bachman, and P. A. Offit. 1987. Rotaviruses isolate W161 representing a presumptive new human serotype. J. Clin. Microbiol. 25:1757-1762[Abstract/Free Full Text].
5. Cubitt, W. D., A. D. Steele, and M. Iturriza. 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[CrossRef][Medline].
6. Cunliffe, N. A., J. S. Gondwe, R. L. Broadhead, M. E. Molyneux, P. A. Woods, J. S. Bresee, R. I. Glass, J. R. Gentsch, and C. A. Hart. 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[CrossRef][Medline].
7. Das, B. K., J. R. Gentsch, H. G. Cicirello, P. A. Woods, A. Gupta, M. Ramachandran, R. Kumar, M. K. Bhan, and R. I. Glass. 1994. Characterization of rotavirus strains from newborns in New Delhi, India. J. Clin. Microbiol. 32:1820-1822[Abstract/Free Full Text].
8. Estes, M. K., and J. Cohen. 1989. Rotavirus gene structure and function. Microbiol. Rev. 53:410-449[Abstract/Free Full Text].
9. Gentsch, J. R., P. A. Woods, M. Ramachandran, B. K. Das, J. P. G. Leite, A. Alfieri, R. Kumar, M. K. Bhan, and R. I. Glass. 1996. Review of G and P typing results from a global collection of rotavirus strains: implications for vaccine development. J. Infect. Dis. 174(Suppl. 1):S30-S36.
10. Gentsch, J. R., R. I. Glass, P. Woods, V. Gouvea, M. Gorziglia, J. Flores, B. K. Das, and M. K. Bhan. 1992. Identification of group A rotavirus gene 4 types by polymerase chain reaction. J. Clin. Microbiol. 30:1365-1373[Abstract/Free Full Text].
11. Gouvea, V., R. I. Glass, P. Woods, K. Taniguchi, H. F. Clark, B. Forrester, and Z. Y. Fang. 1990. Polymerase chain reaction amplification and typing of rotavirus nucleic acid from stool specimens. J. Clin. Microbiol. 28:276-282[Abstract/Free Full Text].
12. Griffin, D. D., C. D. Kirkwood, U. D. Parashar, P. A. Woods, J. S. Bresee, R. I. Glass, and J. R. Gentsch. 2000. Surveillance of rotavirus strains in the United States: identification of unusual strains. J. Clin. Microbiol. 38:2784-2787[Abstract/Free Full Text].
13. Kapikian, A. Z., Y. Hoshino, R. M. Chanock, and I. Pérez-Schael. 1996. Efficacy of a quadrivalent rhesus rotavirus based human rotavirus vaccine aimed at preventing severe rotavirus diarrhea in infants and young children. J. Infect. Dis. 174(Suppl. 1):S65-S72.
14. Leite, J. P. G., A. A. Alfieri, P. A. Woods, R. I. Glass, and J. R. Gentsch. 1996. Rotavirus G and P types circulating in Brazil: characterization by RT-PCR, probe hybridization and sequence analysis. Arch. Virol. 141:2365-2374[CrossRef][Medline].
15. Nakagomi, T., K. Akatani, N. Ikegami, N. Katsushima, and O. Nakagomi. 1988. Occurrence of changes in human rotavirus serotypes with concurrent changes in genomic RNA electropherotypes. J. Clin. Microbiol. 26:2586-2592[Abstract/Free Full Text].
16. O'Halloran, F., M. Lynch, B. Cryan, H. O'Shea, and S. Fanning. 2000. Molecular characterization of rotavirus in Ireland: detection of novel strains circulating in the population. J. Clin. Microbiol. 38:3370-3374[Abstract/Free Full Text].
17. Palombo, E. A., P. J. Masendycz, N. Bogdanovic-Sakran, G. L. Barnes, and R. F. Bishop. 2000. Emergence of serotype G9 human rotaviruses in Australia. J. Clin. Microbiol. 38:1305-1306[Free Full Text].
18. Ramachandran, M., B. K. Das, A. Vij, R. Kumar, S. S. Bhambal, N. Kesari, H. Rawat, L. Bahl, S. Thakur, P. A. Woods, R. I. Glass, M. K. Bhan, and J. R. Gentsch. 1996. Unusual diversity of human rotavirus G and P genotypes in India. J. Clin. Microbiol. 34:436-439[Abstract].
19. Ramachandran, M., J. R. Gentsch, U. D. Parashar, S. Jin, P. A. Woods, J. L. Holmes, C. D. Kirkwood, R. F. Bishop, H. B. Greenberg, S. Urasawa, G. Gerna, B. S. Coulson, K. Tanaguchi, J. S. Bresee, and R. I. Glass. 1998. Detection and characterization of novel rotavirus strains in the United States. J. Clin. Microbiol. 36:3223-3229[Abstract/Free Full Text].
20. Santos, N., R. C. C. Lima, C. F. A. Pereira, and V. Gouvea. 1998. Detection of rotavirus types G8 and G10 among Brazilian children with diarrhea. J. Clin. Microbiol. 36:2727-2729[Abstract/Free Full Text].
21. Unicomb, L. E., G. Podder, J. R. Gentsch, P. A. Woods, K. Z. Hasan, A. S. G. Faruque, M. J. Albert, and R. I. Glass. 1999. Evidence of high-frequency genomic reassortment of group A rotavirus strains in Bangladesh: emergence of type G9 in 1995. J. Clin. Microbiol. 37:1885-1891[Abstract/Free Full Text].
22. Urasawa, S., A. Hasegawa, T. Urasawa, K. Taniguchi, F. Wakasugi, H. Suzuki, S. Inouye, B. Pongprot, J. Supawadee, S. Suprasert, P. Rangsiyanond, S. Tonusin, and Y. Yamazi. 1992. Antigenic and genetic analyses of human rotaviruses in Chiang Mai, Thailand: evidence for a close relationship between human and animal rotaviruses. J. Infect. Dis. 166:227-234[Medline].

Journal of Clinical Microbiology, May 2001, p. 1999-2001, Vol. 39, No. 5
0095-1137/01/$04.00+0   DOI: 10.1128/JCM.39.5.1999-2001.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.


This article has been cited by other articles:

  • Bourdett-Stanziola, L., Jimenez, C., Ortega-Barria, E. (2008). Diversity of Human Rotavirus G and P Genotypes in Panama, Costa Rica, and the Dominican Republic. Am J Trop Med Hyg 79: 921-924 [Abstract] [Full Text]  
  • Araujo, I. T., Heinemann, M. B., Mascarenhas, J. D. P., Assis, R. M. S., Fialho, A. M., Leite, J. P. G. (2007). Molecular analysis of the NSP4 and VP6 genes of rotavirus strains recovered from hospitalized children in Rio de Janeiro, Brazil. J Med Microbiol 56: 854-859 [Abstract] [Full Text]  
  • Mascarenhas, J. D. P., Leite, J. P. G., Lima, J. C., Heinemann, M. B., Oliveira, D. S., Araujo, I. T., Soares, L. S., Gusmao, R. H. P., Gabbay, Y. B., Linhares, A. C. (2007). Detection of a neonatal human rotavirus strain with VP4 and NSP4 genes of porcine origin. J Med Microbiol 56: 524-532 [Abstract] [Full Text]  
  • Lin, Y.-P., Chang, S.-Y., Kao, C.-L., Huang, L.-M., Chung, M.-Y., Yang, J.-Y., Chen, H.-Y., Taniguchi, K., Tsai, K.-S., Lee, C.-N. (2006). Molecular Epidemiology of G9 Rotaviruses in Taiwan between 2000 and 2002.. J. Clin. Microbiol. 44: 3686-3694 [Abstract] [Full Text]  
  • Santos, N., Volotao, E. M., Soares, C. C., Campos, G. S., Sardi, S. I., Hoshino, Y. (2005). Predominance of Rotavirus Genotype G9 during the 1999, 2000, and 2002 Seasons among Hospitalized Children in the City of Salvador, Bahia, Brazil: Implications for Future Vaccine Strategies. J. Clin. Microbiol. 43: 4064-4069 [Abstract] [Full Text]  
  • Banyai, K., Gentsch, J. R., Schipp, R., Jakab, F., Bene, J., Melegh, B., Glass, R. I., Szucs, G. (2004). Molecular epidemiology of human P[8],G9 rotaviruses in Hungary between 1998 and 2001. J Med Microbiol 53: 791-801 [Abstract] [Full Text]  
  • Banyai, K., Gentsch, J. R., Glass, R. I., Uj, M., Mihaly, I., Szucs, G. (2004). Eight-Year Survey of Human Rotavirus Strains Demonstrates Circulation of Unusual G and P Types in Hungary. J. Clin. Microbiol. 42: 393-397 [Abstract] [Full Text]  
  • Kirkwood, C., Bogdanovic-Sakran, N., Palombo, E., Masendycz, P., Bugg, H., Barnes, G., Bishop, R. (2003). Genetic and Antigenic Characterization of Rotavirus Serotype G9 Strains Isolated in Australia between 1997 and 2001. J. Clin. Microbiol. 41: 3649-3654 [Abstract] [Full Text]  
  • Villena, C., El-Senousy, W. M., Abad, F. X., Pinto, R. M., Bosch, A. (2003). Group A Rotavirus in Sewage Samples from Barcelona and Cairo: Emergence of Unusual Genotypes. Appl. Environ. Microbiol. 69: 3919-3923 [Abstract] [Full Text]  
  • Laird, A. R., Gentsch, J. R., Nakagomi, T., Nakagomi, O., Glass, R. I. (2003). Characterization of Serotype G9 Rotavirus Strains Isolated in the United States and India from 1993 to 2001. J. Clin. Microbiol. 41: 3100-3111 [Abstract] [Full Text]  
  • Santos, N., Soares, C. C., Volotao, E. M., Albuquerque, M. C. M., Hoshino, Y. (2003). Surveillance of Rotavirus Strains in Rio de Janeiro, Brazil, from 1997 to 1999. J. Clin. Microbiol. 41: 3399-3402 [Abstract] [Full Text]  
  • Abdel-Haq, N. M., Thomas, R. A., Asmar, B. I., Zacharova, V., Lyman, W. D. (2003). Increased Prevalence of G1P[4] Genotype among Children with Rotavirus-Associated Gastroenteritis in Metropolitan Detroit. J. Clin. Microbiol. 41: 2680-2682 [Abstract] [Full Text]  
  • Rahman, M., De Leener, K., Goegebuer, T., Wollants, E., Van der Donck, I., Hoovels, L. V., Van Ranst, M. (2003). Genetic Characterization of a Novel, Naturally Occurring Recombinant Human G6P[6] Rotavirus. J. Clin. Microbiol. 41: 2088-2095 [Abstract] [Full Text]  
  • Coluchi, N., Munford, V., Manzur, J., Vazquez, C., Escobar, M., Weber, E., Marmol, P., Racz, M. L. (2002). Detection, Subgroup Specificity, and Genotype Diversity of Rotavirus Strains in Children with Acute Diarrhea in Paraguay. J. Clin. Microbiol. 40: 1709-1714 [Abstract] [Full Text]  

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowReprints and Permissions
Right arrow Copyright Information
Right arrow Books from ASM Press
Right arrow MicrobeWorld
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Araújo, I. T.
Right arrow Articles by Leite, J. P. G.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Araújo, I. T.
Right arrow Articles by Leite, J. P. G.

Copyright © 2009 by the American Society for Microbiology.   For an alternate route to Journals.ASM.org, visit: http://intl-journals.asm.org | More Info»