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Journal of Clinical Microbiology, November 2003, p. 5267-5269, Vol. 41, No. 11
0095-1137/03/$08.00+0     DOI: 10.1128/JCM.41.11.5267-5269.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Isolation and Characterization of Dually Reactive Strains of Group A Rotavirus from Hospitalized Children

Sujata S. Ranshing and Shobhana D. Kelkar*

Rotavirus Department, National Institute of Virology, Pune 411 001, India

Received 22 January 2003/ Returned for modification 22 April 2003/ Accepted 13 June 2003


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ABSTRACT
 
Seven rotavirus strains dually reactive to serotype G1- and G2-specific monoclonal antibodies (MAbs) from hospitalized children with rotavirus diarrhea were culture adapted. Six strains were neutralized with G1 antiserum to a higher titer than that of G2, and one was neutralized with G1 and G2 antisera to equal titers. Of these, four strains were also neutralized with G6 antiserum. Five strains with short RNA pattern could not be serotyped, and the remaining two strains with long RNA pattern were serotyped as G1 strains. In addition, two strains showing dual reactivity to G2 and G4 MAbs and one G2-like strain from a nontypeable specimen were isolated. The dual reactivity of the isolates could not be attributed to mixed infections.


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TEXT
 
Surveillance studies on rotaviruses suggest that, in developed countries, 90 to 95% of specimens can be typed as G1 through G4 (9, 17). However, much higher percentages of nontypeable specimens have been reported from developing countries (3, 7, 8, 13, 15, 16, 21, 23, 24). In addition, strains belonging to serotypes G6, G8, and G10 (2, 12, 18, 19, 21) and dually reactive specimens have been detected from India and other developing countries (5, 6, 13, 20, 21). Among the reports from India, the four major worldwide strains made up only 27.4% (10), 65.5% (4), 22.4% (21), and 22% (13) of the total strains by monoclonal antibody (MAb)-based serotyping.

Epidemiological studies carried out in Pune, India, during 1990 to 1997 reported 67 (15.50%) of 432 rotavirus-positive specimens as being dually reactive to serotype-specific MAbs. Twenty-five specimens reacted to G6 and G10 MAbs, 19 specimens reacted to G2 and G4 MAbs, and 13 specimens reacted to G1 and G2 MAbs. The remaining 10 specimens gave dual reactions in several combinations. A large number of specimens (47.92%) remained nontypeable (21). The above data prompted us to study isolation and characterization of some dually reactive and nontypeable fecal specimens, in order to determine whether these specimens really exist in nature or whether the reactivity of original fecal specimens with G1- and G2-specific MAbs was nonspecific in nature. Accordingly, nine dually reactive and three nontypeable specimens from hospitalized children were chosen. The details regarding fecal specimens are presented in Table 1.


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  		TABLE 1. Subgroup, serotype, and RNA pattern of fecal specimens and isolates

Ten isolates were obtained from 12 specimens. Of these, all seven fecal specimens dually reactive to G1 and G2 MAbs yielded isolates. Similarly, rotavirus isolates were obtained from both of the specimens that were dually reactive to G2 and G4 MAbs. Only one specimen (no. 9310361) could be adapted to growth in MA104 cells from three nontypeable specimens. Rotavirus were not isolated from the remaining two fecal specimens in spite of 10 blind passages.

Results of subgrouping, serotyping, and electropherotyping of all the isolates are presented in Table 1. Of 10 isolates, 8 retained the subgroup specificity of their respective original fecal specimens. The subgroup specificity of specimen no. 913700 changed from subgroup I to subgroup I and II after culture adaptation, whereas specimen no. 9016061, which belonged to the non-I, non-II subgroup showed subgroup II specificity. The reason for such a phenomenon is not clear, although it has been reported that fecal specimens having short RNA and belonging to subgroup I and II changed to long RNA with subgroup II upon cultivation (1).

Although strains with short RNA pattern and G2 specificity similar to that of prototype G2 have been reported from India (4, 10, 13, 21), the isolates with dual reactivity having short RNA could not be serotyped. Seventeen strains with subgroup I and short RNA that reacted with G2-specific as well as other serotype-specific MAbs even after adaptation to growth in cell culture have been reported from India (4). Preliminary sequence analyses of the VP7 gene from some of these strains revealed that it was closely related to the corresponding gene of serotype G2 strains of human rotavirus RV5 and S2 but contained point mutations at specific locations. Amino acid changes in the antigenic regions of VP7 may lead to loss of specific reactivity to G2-specific MAbs. Besides, Anand et al. (5) also reported strains exhibiting subgroup I specificity and short RNA that could not be typed as serotype G2 because they showed similar reactivities to MAbs specific for serotypes 1, 2, 3, 4, 6, and 10 (5).

Table 2 summarizes the results of neutralization assays of the isolates with hyperimmune antisera against established prototype rotavirus strains. Six isolates from fecal specimens dually reactive to G1 and G2 MAbs were neutralized with antisera against G1 at a higher titer than with antisera against G2, and one isolate was neutralized with antisera against serotypes G1 and G2 to the same extent. Of the two isolates with dual G2 and G4 specificity, one isolate (no. 938529), with short RNA pattern, was more like G2 than like G4 and the other one (no. 9016061), having a long RNA pattern, was more like G4 than like G2. The isolate no. 9310361, (obtained from the nontypeable specimen) was neutralized more with antiserum against G2 serotype than with antiserum against G1. The results of cross-neutralization of rabbit immune sera against the two isolates no. 958644 and 913700 are presented in Table 3. The antiserum against strain 958644 neutralized serotypes G1 and G2 to an equal extent and neutralized serotype Bov-Lin to higher titers, whereas the antiserum against strain 913700 neutralized the G2 serotype to very high titers. We have taken up studies to raise immune sera and colostrum in pregnant goats against the prototype strains of rotavirus and these two isolates. Both the isolates showed very good neutralizing antibody response to serotypes G1 and G2 in sera as well as colostrum preparation (unpublished data).


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  		TABLE 2. Results of neutralization assays of dually reactive and nontypeable strains


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  		TABLE 3. Results of cross-neutralization assays of immune sera with reference rotavirus strains of different serotypes

Guinea pig erythrocytes were not agglutinated by any of the tissue culture isolates examined. It appears that the isolates are human rotaviruses.

The present study reports cultivation of unusual human rotaviruses from India showing dual serotype specificity for the first time. The results of the study indicate the diversity of rotaviruses in India, as reported earlier (13, 20, 25). Unusual strains related to serotype G1 having subgroup I specificity and short electropherotype have been reported from India (11).

All the isolates and original fecal suspensions reported in the present studies were shown to contain only 11 RNA segments without additional RNA bands. This ruled out the possibility that the patients had mixed infections, although many authors have reported mixed infections by showing the presence of extra RNA bands (3, 14, 26, 27). Thus, the results support the view that a large number of natural reassortants showing dual reactivity may be circulating and affecting children in India. It appears that many nontypeable specimens reported by Kelkar and Ayachit (21) and that belonged to subgroup I with a short RNA pattern could be either G1 or G2 but could not be serotyped. According to Steel et al. (28), dual infections of G1 and G2 may be a result of the predominance of G1 and G2 strains in nature (28).

There are several reports of detection and isolation of dually reactive specimens. Strains dually reactive to G1 and G3 have been detected, (8) and strains reactive to G3 and G5 and to G5 and G11 have been isolated (22, 30, 31). According to Timenetsky et al. (30), one or two amino acid substitutions on G5 VP7 may be sufficient for the G11 reactivity. One potential consequence of the presence of multiple G and/or P types in the specimen is a greater chance for reassortment during natural infections (29). According to Urasawa et al., the prevalence of a variety of human rotavirus strains in a large densely populated area might give rise to simultaneous infections of individuals with more than one human rotavirus strain leading to a gene reassortment among different strains and producing reassortants with unique characters. Following gene reassortment, specimens of dual serotype specificity can occur (31).

In conclusion, it is important to understand antigenic specificities of such reassortants circulating in India in the context of the development of an effective vaccine against rotavirus diarrhea. Moreover, many nontypeable specimens from India having short RNA pattern may be G1 and G2 or G2 and G4, which do not react to MAbs against G2.


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ACKNOWLEDGMENTS
 
Thanks are due to L. B. Bhosale, Technician, Rotavirus Department, NIV, Pune, India, for collection of fecal specimens from hospitalized children.


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FOOTNOTES
 
* Corresponding author. Mailing address: National Institute of Virology, 20-A, Dr. Ambedkar Rd., Pune 411 001, India. Phone: 91-20-6127301. Fax: 91-20-6122669. E-mail: nivrota{at}hotmail.com. Back


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REFERENCES
 
    1
  1. Aboudy, Y., I. Shif, I. Ziberstein, and T. Gotlieb-Stematsky. 1988. Use of polyclonal and monoclonal antibodies and analysis of viral RNA in the detection of unusual group A human rotaviruses. J. Med. Virol. 25:351-359.[Medline]
    2. 2
  2. Adah, M. I., A. Rohwedder, O. D. Olaleyle, and H. Werchau. 1997. Nigerian rotavirus serotype 8 could not be typed by PCR due to nucleotide mutation at the 3' end of the primer binding site. Arch. Virol. 142:1881-1887.[CrossRef][Medline]
    3. 3
  3. Ahmed, M. U., S. Urasawa, K. Taniguchi, N. Kobayashi, F. Wakasugi, A. I. M. M. Islam, and H. A. Shaikh. 1991. Analysis of human rotavirus strains prevailing in Bangladesh in relation to nationwide floods brought by the 1988 monsoon. J. Clin. Microbiol. 29:2273-2279.[Abstract/Free Full Text]
    4. 4
  4. Aijaz, S., K. Gowda, H. V. Jagannath, R. R. Reddy, P. P. Maiya, R. L. Ward, H. B. Greenberg, M. Raju, A. Babu, and C. D. Rao. 1996. Epidemiology of symptomatic human rotaviruses in Bangalore and Mysore, India, from 1988 to 1994 as determined by electropherotype, subgroup and serotype analysis. Arch. Virol. 141:715-726.[CrossRef][Medline]
    5. 5
  5. Anand, T., T. A. Narasa Raju, M. V. Rama Rao, L. Venkateswar Rao, and G. Sharma. 2000. Symptomatic human rotavirus subgroups, serotypes and electropherotypes in Hyderabad, India. Indian J. Med. Res. 112:1-4.[Medline]
    6. 6
  6. Ananthan, S., and P. Saravanan. 1998. Analysis of human rotavirus serotypes in children with acute diarrhoea in Chennai by monoclonal antibody based ELISA. Indian J. Med. Res. 108:58-61.[Medline]
    7. 7
  7. Bern, C., L. Unicomb, J. R. Gentsch, N. Banul, M. Yunus, R. B. Sack, and R. I. Glass. 1992. Rotavirus diarrhea in Bangladeshi children: correlation of disease severity with serotypes. J. Clin. Microbiol. 30:3234-3238.[Abstract/Free Full Text]
    8. 8
  8. Bishop, R. F., L. E. Unicomb, Y. Soenarto, H. Suwardji, Ristanto, and G. L. Barnes. 1989. Rotavirus serotypes causing acute diarrhoea in hospitalized children in Yogyakarta, Indonesia during 1978-1979. Arch. Virol. 107:207-213.[CrossRef][Medline]
    9. 9
  9. Bishop, R. F., L. E. Unicomb, and G. L. Barnes. 1991. Epidemiology of rotavirus serotypes in Melbourne, Australia, 1973 to 1989. J. Clin. Microbiol. 29:862-868.[Abstract/Free Full Text]
    10. 10
  10. Brown, D. W. G., M. M. Mathan, M. M. Mathew, R. Martin, G. M. Beards, and V. I. Mathan. 1988. Rotavirus epidemiology in Vellore, South India: group, subgroup, serotype and electropherotype. J. Clin. Microbiol. 26:2410-2414.[Abstract/Free Full Text]
    11. 11
  11. Chakladar, A., and S. Chakrabarti. 1998. Nucleotide sequence of the VP7 gene of human rotavirus isolated in Calcutta, India: possible emergence of a new subtype of serotype 1. Intervirology 41:127-131.[CrossRef][Medline]
    12. 12
  12. 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]
    13. 13
  13. Das, S., A. Sen, G. Uma, V. Varghese, S. Chaudhuri, S. K. Bhattacharya, T. Krishnan, P. Dutta, D. Dutta, M. K. Bhattacharya, U. Mitra, N. Kobayashi, and T. N. Naik. 2002. Genomic diversity of group A rotavirus strains infecting humans in eastern India. J. Clin. Microbiol. 40:146-149.[Abstract/Free Full Text]
    14. 14
  14. Flores, J., K. Taniguchi, K. Green, I. Perez-Schael, D. Garcia, J. Sears, S. Urasawa, and A. Z. Kapikian. 1988. Relative frequencies of rotavirus serotypes 1, 2, 3, and 4 in Venezuelan infants with gastroenteritis. J. Clin. Microbiol. 26:2092-2095.[Abstract/Free Full Text]
    15. 15
  15. Gentsch, J. R., P. A. Woods, M. Ramachandran, B. K. Das, J. P. Leite, A. Alfrieri, 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.
    16. 16
  16. Georges-Courbot, M. C., A. M. Beraud, G. M. Beards, A. O. Campbell, J. P. Gonzalez, A. S. Georges, and T. H. Flewett. 1988. Subgroups, serotypes and electropherotypes of rotavirus isolated from children in Bangui, Central African Republic. J. Clin. Microbiol. 26:668-671.[Abstract/Free Full Text]
    17. 17
  17. Griffin, D. D., C. D. Kirkwood, U. D. Parashar, P. A. Woods, J. S. Breese, 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]
    18. 18
  18. Holmes, J. L., C. D. Kirkwood, G. Gerna, J. D. Clemens, M. R. Rao, A. B. Naficy, R. Abu-Elyazeed, S. J. Savarino, R. I. Glass, and J. R. Gentsch. 1999. Characterization of unusual G8 rotavirus strains isolated from Egyptian children. Arch. Virol. 144:1381-1396.[CrossRef][Medline]
    19. 19
  19. Jagannath, M. R., R. R. Vethanayagam, Y. B. S. Reddy, S. Raman, and C. D. Rao. 2000. Characterization of human symptomatic rotavirus isolates MP409 and MP480 having 'long' RNA electropherotype and subgroup I specificity, highly related to the P6[1], G8 type bovine rotavirus A5, from Mysore, India. Arch. Virol. 145:1339-1357.[CrossRef][Medline]
    20. 20
  20. Jain, V., B. K. Das, M. K. Bhan, R. I. Glass, J. R. Gentsch, and Indian Strain Surveillance Collaborating Laboratories. 2001. Great diversity of group A rotavirus strains and high prevalence of mixed rotavirus infections in India. J. Clin. Microbiol. 39:3524-3529.[Abstract/Free Full Text]
    21. 21
  21. Kelkar, S. D., and V. L. Ayachit. 2000. Circulation of Group A rotavirus subgroups and serotypes in Pune, India, 1990-1997. J. Health Popul. Nutr. 18:163-170.[Medline]
    22. 22
  22. Nagesha, H. S., J. Huang, C. P. Hum, and H. I. Holmes. 1990. A porcine rotavirus strain with dual VP7 specificity. Virology 175:319-322.[CrossRef][Medline]
    23. 23
  23. Padilla-Noriega, L., C. F. Arias, S. Lopez, F. Puerto, D. R. Snodgrass, K. Taniguchi, and H. B. Greenberg. 1990. Diversity of rotavirus serotypes in Mexican infants with gastroenteritis. J. Clin. Microbiol. 28:1114-1119.[Abstract/Free Full Text]
    24. 24
  24. Pongsuwanne, Y., K. Taniguchi, M. Choonthanom, M. Chiwakul, T. Susansook, S. Saguanwongse, C. Jayavasu, and S. Urasawa. 1989. Subgroup and serotype distributions of human, bovine, and porcine rotavirus in Thailand. J. Clin. Microbiol. 27:1956-1960.[Abstract/Free Full Text]
    25. 25
  25. Ramachandran, M., B. K. Das, A. Vij, R. Kumar, S. S. Bhambai, 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]
    26. 26
  26. Sethi, S. K., D. M. Olive, O. O. Strannegard, and W. Al-Nakib. 1988. Molecular epidemiology of human rotavirus infections based on genome segment variations in viral strains. J. Med. Virol. 26:249-259.[Medline]
    27. 27
  27. Spencer, E. G., L. F. Avindano, and B. I. Garcia. 1983. Analysis of human rotavirus mixed electropherotypes. Infect. Immun. 39:569-572.[Abstract/Free Full Text]
    28. 28
  28. Steel, A. D., M. C. van Niekerk, and M. J. Mphahlele. 1995. Geographic distribution of human rotavirus VP4 genotypes and VP7 serotypes in five South African regions. J. Clin. Microbiol. 33:1516-1519.[Abstract]
    29. 29
  29. Timenetsky, M. do C., N. Santos, and V. Gouvea. 1994. Survey of rotavirus G and P types associated with human gastroenteritis in Sao Paulo, Brazil, from 1986 to 1992. J. Clin. Microbiol. 32:2622-2624.[Abstract/Free Full Text]
    30. 30
  30. Timenetsky, M. do C., V. Gouvea, N. Santos, R. C. C. Carmona, and Y. Hoshino. 1997. A novel human rotavirus serotypes with dual G5-G11 specificity. J. Gen. Virol. 78:1373-1378.[Abstract]
    31. 31
  31. Urasawa, T., S. Urasawa, Y. Chiba, K. Taniguchi, N. Kobayashi, N. L. Mutanda, and P. M. Ptukei. 1987. Antigenic characterization of rotaviruses isolated in Kenya from 1982 to 1983. J. Clin. Microbiol. 25:1891-1896.[Abstract/Free Full Text]

Journal of Clinical Microbiology, November 2003, p. 5267-5269, Vol. 41, No. 11
0095-1137/03/$08.00+0     DOI: 10.1128/JCM.41.11.5267-5269.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.




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