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Distribution of rotavirus VP7 genotypes among children suffering from watery diarrhea in Kolkata, India
Virus Research 87 (2002) 31 – 40 www.elsevier.com/locate/virusres
Distribution of rotavirus VP7 genotypes among children suffering from watery diarrhea in Kolkata, India Dimple Khetawat, Phalguni Dutta, S.K. Bhattacharya, Sekhar Chakrabarti * National Institute of Cholera and Enteric Diseases, P-33 CIT Road, Scheme XM, Beliaghata, Kolkata 700010, India Received 11 December 2001; received in revised form 9 April 2002; accepted 10 April 2002
Abstract A combined reverse transcriptase-polymerase chain reaction (RT/PCR) was used to produce cDNA of the VP7 gene of rotavirus present in the stool samples. A total of 150 rotavirus positive stool samples were used in this study. Multiplex PCR, using the type specific primers, revealed the presence of G1 (49/150, 32.7%), G2 (27/150, 18%) and G4 (30/150, 20%) genotypes among the samples collected during 1999– 2000 from children suffering from acute watery diarrhea. Eighteen samples (12%) were of mixed genotype and the remaining 16 samples (10.6%) could not be typed. Comparative analysis of the full length genes of the representative strains with corresponding genotypes incorporated in the human-rhesus rotavirus tetravalent vaccine (RRV-TV) formulation demonstrates variations of the circulating G1, G2 and G4 strains with the corresponding G genotypes present in the vaccine strain. © 2002 Elsevier Science B.V. All rights reserved. Keywords: Rotavirus; VP7 genes; RT/PCR; Phylogram
Group A rotaviruses have clearly been established as one of the causative agents for severe diarrhea in infants and young children worldwide (Kapikian and Chanock, 1996). The rotavirus genome contains 11 segments of double-stranded RNA surrounded by three capsid layers consisting of a core, an inner capsid and an outer capsid (Estes, 1996b). Antigenic sites used for rotavirus classification are located on VP4 (genome segment 4), VP6 (genome segment 6) and VP7 (genome segment 7, 8 or 9 depending on strain) proteins. A * Corresponding author. Tel.: +91-33-350-4478; fax: + 9133-350-5066 E-mail address: sekhar – [email protected] (S. Chakrabarti).
common feature to all Group A human rotaviruses is the presence of two in-frame initiation codons (nt 49–51 and 136 –138) in VP7, which is generally 1062 bp in length (Kalica et al., 1981). The knowledge of rotavirus serotypes or genotypes seems to play an important role in inducing immunity and subsequent vaccine development (Hoshino and Kapikian, 2000). The typing of rotavirus on the basis of its two outer capsid proteins, VP7 and VP4, has evolved 14 G serotypes (G1 –G14) and 12 P serotypes (Estes, 1996a). G1 –G4 are reported to be predominant globally. Other G types have increasingly been observed in various countries (Beards et al., 1992; Gouvea et al., 1994). Unusual G types associated
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with asymptomatic and symptomatic infections have been reported from various parts of India. Asymptomatic neonates in New Delhi (Ramachandran et al., 1996) were found to be infected by G9 serotype and G10 serotype was detected from Bangalore, a southern city of India (Dunn et al., 1993a). The type-specific monoclonal antibodies were used for serotyping the rotaviruses (Estes, 1996a). However, other typing methods, such as type specific DNA probe-based hybridization (Lin et al., 1987; Larralde and Flores, 1990) and type specific primer-based RT-PCR (Gouvea et al., 1990; Gentsch et al., 1992), have become popular in the recent past. Such methods have been widely used for genotyping rotavirus field isolates and complete concordance between serotype and genotype has so far been observed. During the course of our study for rotavirus surveillance among the hospitalized children suffering from watery diarrhea, we have observed the presence of different VP7 genotypes. In this communication, we report the overall distribution of VP7 genotypes and sequence analysis of few representative samples. Stool samples collected from children who were admitted to Infectious Diseases Hospital and B.C. Roy Children Hospital, Kolkata, India, for the treatment of watery diarrhea, during 1999 – 2000, were screened for rotavirus by ELISA (Krishnan et al., 1994) and polyacrylamide gel electrophoresis of the dsRNA (Laemmli, 1970) extracted from the stool samples. The samples demonstrating the characteristic rotavirus dsRNA pattern (4-2-3-2), (as shown in Fig. 1) in silver-stained polyacrylamide gel (Follet et al., 1984) were selected for
Fig. 1. RNA profile of some of the representative samples.
Table 1 Sequence of primers used in PCR Primer (genotype)
Primer sequence (nucleotide position) (5%–3%)
RV9 RV10
GGCTTTAAAAGAGAGAATT (1–19) GGTCACATCATACAATTCT (1044–1062) CAAGTACTCAAATCAATG (314–331) ATGATATTAACACATTTT (413–430) CGTTTGAAGAAGTTGCAA (689–706) CGTTTCTGGTGAGGAGTT (480–497) CTAGATGTAACTACAACT (757–774)
RV13(G1) RV14(G2) RV15(G3) RV16(G4) RV17(G9)
this study. A total of 150 rotavirus positive samples were used for further characterization. Virus particles were semipurified by ultracentrifugation of fecal sample through 35% (w/v) sucrose at 80,000 g for 1 h at 4 °C. The dsRNA was extracted from the virus pellet and used as template for RT-PCR. Random hexanucleotide primer was used for the synthesis of cDNA which was amplified with antisense, RV10 and sense, RV9 primers to generate full length copies (: 1.1 Kb) of the VP7 gene (Gouvea et al., 1990). Five type-specific primers, RV13–RV17, were pooled and used in combination with the common primer, RV10, in order to produce the specific fragment belonging to G1, G2, G3, G4 and G9 genotypes, respectively (Table 1). The amplification of 748, 649 and 582 bp in the multiplex PCR indicated the prevalence of G1 (49/150, 32.7%), G2 (27/150, 18%) and G4 (30/150, 20%). For 18 samples (12%), dual fragments of different combinations were observed, thus indicating the presence of mixed infection. The remaining 16 samples were found to be rotavirus-positive, but their genotype could not be ascertained using the above primer combinations and thus, they were designated as non-typeable. However, the electrophoretic pattern of these samples (Fig. 1, lane 5) clearly demonstrated the presence of rotavirus in these samples. We could not detect any G9 genotypes in this part of the country, although the presence of G9 was reported from New Delhi, India (Das et al., 1993). One sample from each of the genotype, G1, G2 and G4 was taken for further characterization.
D. Khetawat et al. / Virus Research 87 (2002) 31–40
The full length VP7 gene of these samples (produced by RV9 and RV10 primers) was cloned in the TA cloning vector, pCR™ 2.1 (Invitrogen, USA) following the manufacturer’s protocol. The cloned DNA was sequenced by ABI PRISM method, in the ABI PRISM 310 genetic analyzer, using the M13 forward and reverse primers. The sequences were submitted to the Genbank and the accession numbers are provided at the end of this paper. All the VP7 genes, except one, were found to be 1062 bp in length and possessed two inframe initiation codons. The VP7 gene of one sample, WD33 belonging to G1 genotype, was found to be 1060 bp in length. A deletion of 2 bp at the 5% untranslated region was noticed in this clone. The coding regions (first ATG to TAA) of each clones belonging to the specific genotypes (G1, G2 and G4) were aligned and distance matrix was determined. A divergence of 2– 5% was observed between the clones of same genotypes. The genotype nature of all the strains were further confirmed by comparing the nucleotide sequences and the deduced amino acid sequences with reference strains, G1– G14. The University of Wisconsin Genetics Computer Group (GCG) program was used to pile up and compare these VP7 genes with corresponding gene of all the serotypes from G1 to G14. The percentage homology with the reference strains is given in Table 2, along with the accession number of the reference strains. The homology in the VP7 gene between WD33 strain and strains of G2– G14 was 72– 76% and 76– 89% in nucleotide and amino acid sequences, respectively. In contrast, WD33 was 94% and 97% homologous to the G1 strain, Wa in nucleotide and amino acid sequences, respectively. The homology in the VP7 gene between SC4 strain and strains of G1– G14, barring G2 was 73 – 77% and 77–82% in nucleotide and predicted amino acid sequences, respectively. In contrast, SC4 was 97 and 98% homologous to the S2 strain (G2 serotype) in nucleotide and amino acid sequences, respectively. The homology in the VP7 gene between SC134 strain and strains of G1– G14, barring G4 was 71–77% and 76–82% in nucleotide and predicted amino acid sequences, respectively. In contrast, SC134 was 97 and 97.2% homologous to the ST3
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strain (G4 serotype, subtype ‘A’), 87 and 95% homologous to the PV5257 strain (G4 serotype, subtype ‘B’) and 88 and 96% homologous to the Gottfried strain (G4 serotype, porcine) in nucleotide and amino acid sequences, respectively. One representative from each genotype was taken for the phylogenetic analysis. Phylip3.5 computer programs (Devereaux et al., 1984; Felsenstein, 1989) were used to examine the relatedness of local strain to the other strains of the same genotype. A distance matrix table was prepared from amino acid sequences deduced from the VP7 open reading frame. Distance matrices for deduced VP7 amino acid sequences used to generate phylogenetic trees were calculated with the PROTDIST program using the Kimura protein method. The evolutionary trees presented in this work (Fig. 2) were prepared with the Kitsch module of the Phylip 3.5 software programs using a Neighbor–Joining method (Felsenstein, 1989). The names of the isolates and their corresponding accession numbers are shown in Table 3. In the phylogenetic analysis, amino acid sequence of the VP7 gene of the WD33 (G1 strain) did not match with the prototype G1 strain, Wa. More interestingly, this strain was found not to be related to any of the strains; but, only distantly related with the Egyptian strain, egy-7, although the two strains differed from each other as revealed by the distance separating them (Fig. 2A). Phylogenetic analysis of the global G2 strains revealed two clusters (Fig. 2B) (Wen et al., 1995; Piec and Palambo, 1998). Geographical clustering was very much apparent from the phylogenetic tree. The strain, M48, isolated from Manipur, a north eastern state of India clustered with the Kolkata strain, SC4, though they were isolated 10 years apart. VP7 of DS-1 strain, which is incorporated into the tetravalent rhesus rotavirus vaccine formulation, seemed to be dissimilar to the circulating G2 rotavirus strain (SC4) detected in Kolkata, India. The local G4 strain SC134 clustered with the Italian strain, PV5249 and the South African strain, GR846/86, (Fig. 2C) belonging to G4 genotype. The analysis shows that SC134 is related to prototype ST3 strain though there is a horizontal distance separating the two.
PV5257 (M86832) OSU (X04613) NCDV (M12394) Ch2 (X56784) B37 (J04334) E116 (L14072) B223 (X52650) YM (M23194) L26 (M36396) L338 (D00843) F123 (M61876)
G1 94 G2 73 G3 75 G4 77 G4 (Subtype A) G4 (Subtype B) G5 76 G6 75 G7 64 G8 72 G9 76 G10 75 G11 75 G12 74 G13 74 G14 75
Wa (K02033) S2 (M11164) SA11 (V01546) Gottfried (X06759) ST3 (X13603)
89 87 76 84 88 87 87 87 85 88
97 86 89 87
74 73 73 73 75 74 77 74 74 74
75 97 75 73
Nucleotide sequence
Nucleotide sequence
Amino acid sequence
SC4
WD33
% Homology with VP7 of strain
G serotype
Strain (accession No.)
81 80 80 79 81 80 82 82 79 80
82 98 81 77
Amino acid sequence
87 77 74 73 71 75 73 76 73 73 74
76 72 75 88 97
Nucleotide sequence
SC134
95 81 82 79 77 81 80 81 80 80 82
81 76 83 96 97
Amino acid sequence
Table 2 Comparison of nucleotide and predicted amino acid sequences of the VP7 gene of rotavirus with corresponding genes of other rotavirus G types
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D. Khetawat et al. / Virus Research 87 (2002) 31–40
Amino acid alignment of the representative strains was carried out with the corresponding reference strain, as shown in Fig. 3. The predicted amino acid sequence of WD33 was compared with prototype G1 strain, Wa. The highest degree of homology in the deduced amino acid sequences were found in the VR5 (93%) and VR8 (94%) region with that of prototype G1 strain, Wa. Further analysis (Fig. 3A) revealed that there were 16 amino acid changes in total, of which nine were in the variable domains, one each in VR5, VR7 and VR8. In VR3, there were two and in VR4, four changes were present. The homology of the local G2 strain, SC4, with the prototype S2 strain (G2) was found to be 85.71% in VR5 region. The amino acid homology in the VR8 region was more profound (100%). Amino acid alignment (Fig. 3B) revealed only five changes in SC4 with respect to the prototype strain, S2. The changes were one each in VR3 and VR9 and two in VR5. One was outside the variable domain. In antigenic region A (87– 101), the alanine (A) in DS-1 was replaced by threonine (T)
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at 87. Antigenic regions B and C were similar to DS-1. These are sites that have been shown to be involved in the neutralization of serotype G2 viruses by N-MAbs (Lazdins et al., 1995). Overall, there was a difference of 12 amino acids between the local strain SC134 and the prototype strain, ST3 (Fig. 3C). Out of this, one each was present in regions VR1, VR4 and VR6. In VR7, there were three and in VR9, there were two. No changes were there in VR2, VR3, VR5 and VR8. Four of the changes were outside the variable domains. The VP7 glycoprotein (34–38 kDa in size) is the major neutralization antigen of rotaviruses detected by hyperimmune antiserum and serves as the basis for determination of serotype (Bastardo et al., 1981; Killen and Dimmock, 1982; Lazdins et al., 1985; Sonza et al., 1984). Comparison of the VP7 of strains belonging to 14 rotavirus serotypes indicated that there are nine regions viz. VR1(aa9–20), VR2(25–32), VR3(37–53), VR4(65– 76) VR5(87– 100), VR6(119–132), VR7(141–150), VR8(208–224) and VR9(235–
Fig. 2. Phylogram of VP7 proteins deduced from local strains, WD33 (G1), SC4 (G2) and SC134 (G4). The strains, d, DS1 and ST3 were used for the human-rhesus rotavirus tetravalent vaccine formulation. The phylogenetic tree was generated from a VP7 amino acid multiple sequence file using the Prodist and Kitsch modules of the Phylip 3.5 program to calculate a distance matrix and draw the tree.
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Fig. 3. Comparison of the predicted amino acid sequence of WD33, SC4 and SC134 with that of the respective reference strains, Wa, S2 and ST3.The positions of the variable domains, VR1-VR9 are indicated.
D. Khetawat et al. / Virus Research 87 (2002) 31–40
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Table 3 List of strains used in the construction of the phylogenetic trees along with the respective accession numbers Serial No.
G1 isolates (accession no.)
G2 isolates (accession no.)
G4 isolates (accession no.)
1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30. 31. 32. 33. 34. 35. 36. 37. 38. 39. 40. 41.
brz-2 (U26362) brz-3 (U26363) ban-48 (U26364) brz-4 (U26365) ban-59 (U26366) brz-5 (U26367) brz-6 (U26368) cos-69 (U26369) cos-70 (U26370) chi-45 (U26371) chi-46 (U26372) egy-7 (U26373) egy-8 (U26374) isr-55 (U26375) isr-56 (U26376) kor-54 (U26377) kor-64 (U26378) wadc-6 (U26379) md-216 (U26380) mo-12 (U26381) mo-15 (U26382) neb-49 (U26383) ohio-11 (U26384) ohio-25 (U26385) ohio-40 (U26386) ohio-64 (U26387) ri-14 (U26388) ri-39 (U26389) ri-43 (U26390) rus-91 (U26391) tn-16 (U26392) tn-39 (U26393) va-12 (U26394) zam-52 (U26395) jpn-417 (D16328) jpn-418 (D16327) jpn-421 (D16326) Wa (K02033) d (M37340) aus-91 (M93006) WD33 (Y18786)
1992A (U73947) 1992B (U73948) 1992C (U73949) 1994A (U73950) 1994B (U73952) 1994C (U73951) 1994D (U73953) 1994E (U73954) 1995A (U73955) 1995B (U73956) 1995C (U73957) 1995D (U73958) CHIN1 (D50112) CHIN3 (D50113) CHIN5 (D50114) CHIN7 (D50115) CHIN8 (D50116) PAK458 (D50126) PAK426 (D50125) JAPAN076 (D50119) JAPAN038 (D50118) JAPAN0022 (D50117) JAPAN085 (D50120) JAPAN137 (D50121) JAPAN21 (D50122) JAPAN58 (D50123) DS1 (M37348) S2 (M11164) KUN (D50124) TMC-II (D50127) RV5 (M28377) HN126 (M37349) TW3780 (AF044348) TF85 (AF106299) TA26 (AF106284) M48 (L11605) SC4 (AJ293718)
Rota 104 (AB012065) Rota 231 (AB012066) Rota 349 (AB012067) Rota 399 (AB012068) Rota 49 (AB012069) Rota 39 (AB012070) Rota 513 (AB012071) Rota 544 (AB012072) Rota 551 (AB012073) Rota 554 (AB012074) Rota 106 (AB012075) Rota 3 (AB012076) Rota 331 (AB012077) Rota Hochi (AB012078) Rota Odelia (AB012079) GR442/86 (AF161817) GR630/86 (AF161818) NB111/86 (AF161819) NB187/86 (AF161820) NB123/86 (AF161821) GR846/86 (AF161822) GR1107/86 (AF161823) GR828/86 (AF170834) GR833/86 (AF170835) GR856/86 (AF170836) GR1106/86 (AF170837) ST3 (X13603) PV5249 (M86490) VA79 (M86834) PV5257 (M86832) VA75 (M86833) SC134 (AJ278217)
242), of the linear amino acid sequence of VP7 which are highly divergent (Green et al., 1989). Each of these regions is highly conserved in human rotavirus strains within the same serotype. There is sufficient conservation of sequence of human rotavirus strains within a serotype that it is possible to predict the serotype of an isolate by
direct sequence analysis of two of these variable regions (VR5 and VR8) of VP7 (Green et al., 1987, 1988, 1989). Immunological studies using neutralizing monoclonal antibodies (N-MAbs) directed at VP7 have been classified into three functional groups: monotype-specific, serotype-specific and cross-reactive. Studies involving generation of
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mutants resistant to neutralization by N-MAbs and sequencing of the gene encoding VP7 of such mutants indicated that the VR5 (region A, amino acids 87–96) is involved in monotype specific, serotype specific or cross-reactive neutralization and both VR7 (region B, amino acids 145– 150) and VR8 (region C, amino acids 211– 223) contain monotype-specific or serotype specific neutralization sites (Dyall-Smith et al., 1986; Mackow et al., 1988; Taniguchi et al., 1988; Matsui et al., 1989; Coulson and Kirkwood, 1991; Kobayashi et al., 1991a,b). These regions exhibit clustered amino acid variation, most notably between rotaviruses of different G type (Dyall-Smith et al., 1986; Green et al., 1987, 1988). Mutations in the A and C regions have been reported in variants of G1 viruses Ku (Taniguchi et al., 1988; Kobayashi et al., 1991a), RV-4 (Coulson and Kirkwood, 1991) and K8 (Kobayashi et al., 1991b). Mutations in these regions were also detected in variants of the G2 virus DS-1 (Dunn et al., 1993b), G3 viruses (Mackow et al., 1988; Hoshino et al., 1994), G9 viruses (Kirkwood et al., 1993) and G4, 6, 9 and 10 viruses (Hoshino et al., 1994). It seems possible that the circulating G1, G2 and G4 strains may have significant differences from ‘D’, ‘DS1’ and ‘ST3’ in their VP7 gene sequences and this, in turn, may cause significant differences in anti-D, DS1 and ST3 neutralizing antibody titers found in vaccinees relative to the titers of the circulating strains examined. From our study, it is apparent that G1, G2 and G4 genotypes of rotavirus are, at present, prevalent in this part of the country. The phylogenetic analysis of representative strains demonstrates the relation with the genotypes incorporated in the rhesus-rotavirus tetravalent vaccine formulation. The first human rotavirus vaccine (a human-animal tetravalent reassortant virus mixture) was licensed in the US in 1998 (Parashar et al., 1998). The human-rhesus rotavirus reassortant vaccines were developed with the goal of combining the specificity of the epidemiologically important VP7 serotypes with the attenuation phenotype of RRV, MMU18006 (Midthun et al., 1985, 1986). Human-RRV reassortant strains were recovered after coinfection of AGMK cell cultures with the RRV vaccine strain, MMU18006 and human ro-
tavirus strain D, DS-1 or ST3. The D (VP7 serotype 1) and DS-1 (VP7 serotype 2) strains were initially detected in stools of children hospitalized with diarrhea and were then passaged in gnotobiotic calves, whereas, ST3 (VP7 serotype 4) was derived from the stool of an asymptomatic new born infant and was isolated in AGMK cells. Vaccine lots were subsequently prepared in DBSFRhL2 cells, a semi-continuous diploid cell strain developed as a suitable vaccine substrate. The efficacy of this vaccine in developing countries has been variable, but, in a recent trial involving more than 2200 underprivileged urban children in Venezuela, vaccine efficacy approached levels seen in industrialized countries (48% against all episodes of diarrhea, 70% against episodes requiring hospitalization, 75% against dehydrating illness and 88% against severe episodes of rotavirus diarrhea) (Perez-Schael et al., 1997). A better understanding of the population genetics of rotavirus will assist in the development of more effective vaccine formulations and strategies. The information on rotavirus genotypes/serotypes seems to play an important role in inducing immunity and subsequent vaccine development (Jin et al., 1996; Gentsch et al., 1996; Hoshino and Kapikian, 2000). Variation within a genotype is well documented and subtypes within genotype has also been described (Palombo et al., 1993). Strains of less common or unusual serotypes, which also cause diarrhea have been described and constitute 14% of global isolates. In particular, recent surveys carried out in Brazil, India and Bangladesh, demonstrated the emergence of serotypes G5 and G9 that are not covered by the RRV-TV vaccine (Kapikian and Chanock, 1996). Again, difference in antigenic responses has also been reported, based on the genetic variation (Coulson et al., 1996; Diwakarla and Palombo, 1999). In particular, variation is seen in VP7, the viral protein targeted by current vaccine strategies. Ultimately, the effect that diversity of the viral population has on the success of human rotavirus vaccines and the role that mass vaccination will have on rotavirus evolution, should be determined from surveillance and epidemiological studies of clinical isolates before and after the introduction of vaccination programs.
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Acknowledgements We thank Dr Jon R. Gentsch, Viral Gastroenteritis Section, Center for Disease Control and Prevention, Atlanta, GA, for his advice and critical comments. The sequences of the VP7 genes used in this study were submitted to the Genbank and the assigned Accession Nos. are Y18786 (WD33), AJ293718 (SC4) and AJ278217 (SC134).
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Genes of human (strain Wa) and bovine (strain UK) rotaviruses that code for neutralization and subgroup antigens. Virology 112, 385 –390. Kapikian, A.Z., Chanock, R.M., 1996. Rotaviruses. In: Fields, B.N., Knipe, D.M., Howley, P.M., Chanock, R.M., Melnick, J.L., Monath, T.P., Roizman, B., Straus, S.E. (Eds.), Fields Virology, third ed. Lippincott-Raven, Philadelphia, pp. 1657 – 1708. Killen, H.M., Dimmock, N.J., 1982. Identification of a neutralization-specific antigen of a calf rotavirus. J. Gen. Virol. 62 (Pt 2), 297 –311. Kirkwood, C., Masendycz, P.J., Coulson, B.S., 1993. Characteristics and location of cross-reactive and serotype-specific neutralization sites on VP7 of human G type 9 rotaviruses. Virology 196, 79 – 88. Kobayashi, N., Taniguchi, K., Urasawa, S., 1991a. Analysis of the newly identified neutralization epitopes on VP7 of human rotavirus serotype 1. J. Gen. Virol. 72, 117 –124. Kobayashi, N., Taniguchi, K., Urasawa, T., Urasawa, S., 1991b. Analysis of the neutralization epitopes on human rotavirus VP7 recognized by monotype-specific monoclonal antibodies. J. Gen. Virol. 72 (Pt 8), 1855 –1861. Krishnan, T., Burke, B., Shen, S., Naik, T.N., Desselberger, U., 1994. Molecular epidemiology of human rotavirus in Manipur: genome analysis of rotavirus of long electropherotype and subgroup I. Arch. Virol. 134, 279 –292. Laemmli, U.K., 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680 – 685. Larralde, G., Flores, J., 1990. Identification of gene 4 alleles among human rotaviruses by polymerase chain reactionderived probes. Virology 179, 469 –473. Lazdins, I., Sonza, S., Dyall-Smith, M.L., Coulson, B.S., Holmes, I.H., 1985. Demonstration of an immunodominant neutralization site by analysis of antigenic variants of SA11 rotavirus. J. Virol. 56, 317 –319. Lazdins, I., Coulson, B.S., Kirkwood, C., Dyall-Smith, M., Masendycz, P.J., Sonza, S., Holmes, I.H., 1995. Rotavirus antigenicity is affected by the genetic context and glycosylation of VP7. Virology 209, 80 –89. Lin, M., Imai, M., Ikegami, N., Bellamy, A.R., Summers, D., Nuss, D.L., Deibel, R., Furuichi, Y., 1987. cDNA probes of individual genes of human rotavirus distinguish viral subgroups and serotypes. J. Virol. Methods 15, 285 – 289. Mackow, E.R., Shaw, R.D., Matsui, S.M., Vo, P.T., Benfield, D.A., Greenberg, H.B., 1988. Characterization of homotypic and heterotypic VP7 neutralization sites of rhesus
rotavirus. Virology 165, 511 – 517. Matsui, S.M., Mackow, E.R., Greenberg, H.B., 1989. Molecular determinants of rotavirus neutralization and protection. Adv. Virus Res. 36, 181 – 214. Midthun, K., Greenberg, H.B., Hoshino, Y., Kapikian, A.Z., Wyatt, R.G., Chanock, R.M., 1985. Reassortant rotaviruses as potential live rotavirus vaccine candidates. J. Virol. 53, 949 – 954. Midthun, K., Hoshino, Y., Kapikian, A.Z., Chanock, R.M., 1986. Single gene substitution rotavirus reassortants containing the major neutralization protein (VP7) of human rotavirus serotype 4. J. Clin. Microbiol. 24, 822 – 826. Palombo, E.A., Bishop, R.F., Cotton, R.G.H., 1993. Sequence conservation within neutralization epitope regions of VP7 and VP4 proteins of human serotype G4 rotavirus isolates. Arch. Virol. 142, 1481 – 1489. Parashar, U.D., Breese, J.S., Gentsch, J.R., Glass, R.I., 1998. Rotavirus. Emerg. Infect. Dis. 4, 561 – 570. Perez-Schael, I., Guntinas, M.J., Perez, M., Pagone, V., Rojas, A.M., Gonzlez, R., Cunto, W., Hoshino, Y., Kapikian, A.Z., 1997. Efficacy of the rhesus-rotavirus based quadrivalent vaccine in infants and young children in Venezuela. New Engl. J. Med. 337, 1181 – 1187. Piec, T.L., Palambo, E.A., 1998. Sequence comparison of the VP7 of serotype G2 rotaviruses from diverse geographical locations. DNA Seq. 9, 369 – 373. Ramachandran, M., Das, B.K., Vij, A., Kumar, R., Bhambal, S.S., Kesari, N., Rawat, H., Bhal, L., Thakur, S., Woods, P.A., Glass, R.I., Bhan, M.K., Gentsch, J.R., 1996. Unusual diversity of human rotavirus G and P genotypes in India. J. Clin. Microbiol. 34, 436 – 439. Sonza, S., Breschkin, A.M., Holmes, I.H., 1984. The major surface glycoprotein of simian rotavirus (SA11) contains distinct epitopes. Virology 134 (2), 318 – 327. Taniguchi, K., Hoshino, Y., Nishikawa, K., Green, K.Y., Maloy, W.L., Morita, Y., Urasawa, S., Kapikian, A.Z., Chanock, R.M., Gorziglia, M., 1988. Cross-reactive and serotype-specific neutralization epitopes on VP7 of human rotavirus: nucleotide sequence analysis of antigenic mutants selected with monoclonal antibodies. J. Virol. 62, 1870 – 1874. Wen, L., Ushijima, H., Kakizawa, J., Fang, Z., Nishio, O., Morikawa, S., Motohiro, T., 1995. Genetic variation in VP7 gene of human rotavirus serotype 2 (G2 type) isolated in Japan, China, and Pakistan. Microbiol. Immunol. 39, 911 – 915.
Distribution of rotavirus VP7 genotypes among children suffering from watery diarrhea in Kolkata, India Dimple Khetawat, Phalguni Dutta, S.K. Bhattacharya, Sekhar Chakrabarti * National Institute of Cholera and Enteric Diseases, P-33 CIT Road, Scheme XM, Beliaghata, Kolkata 700010, India Received 11 December 2001; received in revised form 9 April 2002; accepted 10 April 2002
Abstract A combined reverse transcriptase-polymerase chain reaction (RT/PCR) was used to produce cDNA of the VP7 gene of rotavirus present in the stool samples. A total of 150 rotavirus positive stool samples were used in this study. Multiplex PCR, using the type specific primers, revealed the presence of G1 (49/150, 32.7%), G2 (27/150, 18%) and G4 (30/150, 20%) genotypes among the samples collected during 1999– 2000 from children suffering from acute watery diarrhea. Eighteen samples (12%) were of mixed genotype and the remaining 16 samples (10.6%) could not be typed. Comparative analysis of the full length genes of the representative strains with corresponding genotypes incorporated in the human-rhesus rotavirus tetravalent vaccine (RRV-TV) formulation demonstrates variations of the circulating G1, G2 and G4 strains with the corresponding G genotypes present in the vaccine strain. © 2002 Elsevier Science B.V. All rights reserved. Keywords: Rotavirus; VP7 genes; RT/PCR; Phylogram
Group A rotaviruses have clearly been established as one of the causative agents for severe diarrhea in infants and young children worldwide (Kapikian and Chanock, 1996). The rotavirus genome contains 11 segments of double-stranded RNA surrounded by three capsid layers consisting of a core, an inner capsid and an outer capsid (Estes, 1996b). Antigenic sites used for rotavirus classification are located on VP4 (genome segment 4), VP6 (genome segment 6) and VP7 (genome segment 7, 8 or 9 depending on strain) proteins. A * Corresponding author. Tel.: +91-33-350-4478; fax: + 9133-350-5066 E-mail address: sekhar – [email protected] (S. Chakrabarti).
common feature to all Group A human rotaviruses is the presence of two in-frame initiation codons (nt 49–51 and 136 –138) in VP7, which is generally 1062 bp in length (Kalica et al., 1981). The knowledge of rotavirus serotypes or genotypes seems to play an important role in inducing immunity and subsequent vaccine development (Hoshino and Kapikian, 2000). The typing of rotavirus on the basis of its two outer capsid proteins, VP7 and VP4, has evolved 14 G serotypes (G1 –G14) and 12 P serotypes (Estes, 1996a). G1 –G4 are reported to be predominant globally. Other G types have increasingly been observed in various countries (Beards et al., 1992; Gouvea et al., 1994). Unusual G types associated
0168-1702/02/$ - see front matter © 2002 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 8 - 1 7 0 2 ( 0 2 ) 0 0 0 8 1 - 3
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with asymptomatic and symptomatic infections have been reported from various parts of India. Asymptomatic neonates in New Delhi (Ramachandran et al., 1996) were found to be infected by G9 serotype and G10 serotype was detected from Bangalore, a southern city of India (Dunn et al., 1993a). The type-specific monoclonal antibodies were used for serotyping the rotaviruses (Estes, 1996a). However, other typing methods, such as type specific DNA probe-based hybridization (Lin et al., 1987; Larralde and Flores, 1990) and type specific primer-based RT-PCR (Gouvea et al., 1990; Gentsch et al., 1992), have become popular in the recent past. Such methods have been widely used for genotyping rotavirus field isolates and complete concordance between serotype and genotype has so far been observed. During the course of our study for rotavirus surveillance among the hospitalized children suffering from watery diarrhea, we have observed the presence of different VP7 genotypes. In this communication, we report the overall distribution of VP7 genotypes and sequence analysis of few representative samples. Stool samples collected from children who were admitted to Infectious Diseases Hospital and B.C. Roy Children Hospital, Kolkata, India, for the treatment of watery diarrhea, during 1999 – 2000, were screened for rotavirus by ELISA (Krishnan et al., 1994) and polyacrylamide gel electrophoresis of the dsRNA (Laemmli, 1970) extracted from the stool samples. The samples demonstrating the characteristic rotavirus dsRNA pattern (4-2-3-2), (as shown in Fig. 1) in silver-stained polyacrylamide gel (Follet et al., 1984) were selected for
Fig. 1. RNA profile of some of the representative samples.
Table 1 Sequence of primers used in PCR Primer (genotype)
Primer sequence (nucleotide position) (5%–3%)
RV9 RV10
GGCTTTAAAAGAGAGAATT (1–19) GGTCACATCATACAATTCT (1044–1062) CAAGTACTCAAATCAATG (314–331) ATGATATTAACACATTTT (413–430) CGTTTGAAGAAGTTGCAA (689–706) CGTTTCTGGTGAGGAGTT (480–497) CTAGATGTAACTACAACT (757–774)
RV13(G1) RV14(G2) RV15(G3) RV16(G4) RV17(G9)
this study. A total of 150 rotavirus positive samples were used for further characterization. Virus particles were semipurified by ultracentrifugation of fecal sample through 35% (w/v) sucrose at 80,000 g for 1 h at 4 °C. The dsRNA was extracted from the virus pellet and used as template for RT-PCR. Random hexanucleotide primer was used for the synthesis of cDNA which was amplified with antisense, RV10 and sense, RV9 primers to generate full length copies (: 1.1 Kb) of the VP7 gene (Gouvea et al., 1990). Five type-specific primers, RV13–RV17, were pooled and used in combination with the common primer, RV10, in order to produce the specific fragment belonging to G1, G2, G3, G4 and G9 genotypes, respectively (Table 1). The amplification of 748, 649 and 582 bp in the multiplex PCR indicated the prevalence of G1 (49/150, 32.7%), G2 (27/150, 18%) and G4 (30/150, 20%). For 18 samples (12%), dual fragments of different combinations were observed, thus indicating the presence of mixed infection. The remaining 16 samples were found to be rotavirus-positive, but their genotype could not be ascertained using the above primer combinations and thus, they were designated as non-typeable. However, the electrophoretic pattern of these samples (Fig. 1, lane 5) clearly demonstrated the presence of rotavirus in these samples. We could not detect any G9 genotypes in this part of the country, although the presence of G9 was reported from New Delhi, India (Das et al., 1993). One sample from each of the genotype, G1, G2 and G4 was taken for further characterization.
D. Khetawat et al. / Virus Research 87 (2002) 31–40
The full length VP7 gene of these samples (produced by RV9 and RV10 primers) was cloned in the TA cloning vector, pCR™ 2.1 (Invitrogen, USA) following the manufacturer’s protocol. The cloned DNA was sequenced by ABI PRISM method, in the ABI PRISM 310 genetic analyzer, using the M13 forward and reverse primers. The sequences were submitted to the Genbank and the accession numbers are provided at the end of this paper. All the VP7 genes, except one, were found to be 1062 bp in length and possessed two inframe initiation codons. The VP7 gene of one sample, WD33 belonging to G1 genotype, was found to be 1060 bp in length. A deletion of 2 bp at the 5% untranslated region was noticed in this clone. The coding regions (first ATG to TAA) of each clones belonging to the specific genotypes (G1, G2 and G4) were aligned and distance matrix was determined. A divergence of 2– 5% was observed between the clones of same genotypes. The genotype nature of all the strains were further confirmed by comparing the nucleotide sequences and the deduced amino acid sequences with reference strains, G1– G14. The University of Wisconsin Genetics Computer Group (GCG) program was used to pile up and compare these VP7 genes with corresponding gene of all the serotypes from G1 to G14. The percentage homology with the reference strains is given in Table 2, along with the accession number of the reference strains. The homology in the VP7 gene between WD33 strain and strains of G2– G14 was 72– 76% and 76– 89% in nucleotide and amino acid sequences, respectively. In contrast, WD33 was 94% and 97% homologous to the G1 strain, Wa in nucleotide and amino acid sequences, respectively. The homology in the VP7 gene between SC4 strain and strains of G1– G14, barring G2 was 73 – 77% and 77–82% in nucleotide and predicted amino acid sequences, respectively. In contrast, SC4 was 97 and 98% homologous to the S2 strain (G2 serotype) in nucleotide and amino acid sequences, respectively. The homology in the VP7 gene between SC134 strain and strains of G1– G14, barring G4 was 71–77% and 76–82% in nucleotide and predicted amino acid sequences, respectively. In contrast, SC134 was 97 and 97.2% homologous to the ST3
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strain (G4 serotype, subtype ‘A’), 87 and 95% homologous to the PV5257 strain (G4 serotype, subtype ‘B’) and 88 and 96% homologous to the Gottfried strain (G4 serotype, porcine) in nucleotide and amino acid sequences, respectively. One representative from each genotype was taken for the phylogenetic analysis. Phylip3.5 computer programs (Devereaux et al., 1984; Felsenstein, 1989) were used to examine the relatedness of local strain to the other strains of the same genotype. A distance matrix table was prepared from amino acid sequences deduced from the VP7 open reading frame. Distance matrices for deduced VP7 amino acid sequences used to generate phylogenetic trees were calculated with the PROTDIST program using the Kimura protein method. The evolutionary trees presented in this work (Fig. 2) were prepared with the Kitsch module of the Phylip 3.5 software programs using a Neighbor–Joining method (Felsenstein, 1989). The names of the isolates and their corresponding accession numbers are shown in Table 3. In the phylogenetic analysis, amino acid sequence of the VP7 gene of the WD33 (G1 strain) did not match with the prototype G1 strain, Wa. More interestingly, this strain was found not to be related to any of the strains; but, only distantly related with the Egyptian strain, egy-7, although the two strains differed from each other as revealed by the distance separating them (Fig. 2A). Phylogenetic analysis of the global G2 strains revealed two clusters (Fig. 2B) (Wen et al., 1995; Piec and Palambo, 1998). Geographical clustering was very much apparent from the phylogenetic tree. The strain, M48, isolated from Manipur, a north eastern state of India clustered with the Kolkata strain, SC4, though they were isolated 10 years apart. VP7 of DS-1 strain, which is incorporated into the tetravalent rhesus rotavirus vaccine formulation, seemed to be dissimilar to the circulating G2 rotavirus strain (SC4) detected in Kolkata, India. The local G4 strain SC134 clustered with the Italian strain, PV5249 and the South African strain, GR846/86, (Fig. 2C) belonging to G4 genotype. The analysis shows that SC134 is related to prototype ST3 strain though there is a horizontal distance separating the two.
PV5257 (M86832) OSU (X04613) NCDV (M12394) Ch2 (X56784) B37 (J04334) E116 (L14072) B223 (X52650) YM (M23194) L26 (M36396) L338 (D00843) F123 (M61876)
G1 94 G2 73 G3 75 G4 77 G4 (Subtype A) G4 (Subtype B) G5 76 G6 75 G7 64 G8 72 G9 76 G10 75 G11 75 G12 74 G13 74 G14 75
Wa (K02033) S2 (M11164) SA11 (V01546) Gottfried (X06759) ST3 (X13603)
89 87 76 84 88 87 87 87 85 88
97 86 89 87
74 73 73 73 75 74 77 74 74 74
75 97 75 73
Nucleotide sequence
Nucleotide sequence
Amino acid sequence
SC4
WD33
% Homology with VP7 of strain
G serotype
Strain (accession No.)
81 80 80 79 81 80 82 82 79 80
82 98 81 77
Amino acid sequence
87 77 74 73 71 75 73 76 73 73 74
76 72 75 88 97
Nucleotide sequence
SC134
95 81 82 79 77 81 80 81 80 80 82
81 76 83 96 97
Amino acid sequence
Table 2 Comparison of nucleotide and predicted amino acid sequences of the VP7 gene of rotavirus with corresponding genes of other rotavirus G types
34 D. Khetawat et al. / Virus Research 87 (2002) 31–40
D. Khetawat et al. / Virus Research 87 (2002) 31–40
Amino acid alignment of the representative strains was carried out with the corresponding reference strain, as shown in Fig. 3. The predicted amino acid sequence of WD33 was compared with prototype G1 strain, Wa. The highest degree of homology in the deduced amino acid sequences were found in the VR5 (93%) and VR8 (94%) region with that of prototype G1 strain, Wa. Further analysis (Fig. 3A) revealed that there were 16 amino acid changes in total, of which nine were in the variable domains, one each in VR5, VR7 and VR8. In VR3, there were two and in VR4, four changes were present. The homology of the local G2 strain, SC4, with the prototype S2 strain (G2) was found to be 85.71% in VR5 region. The amino acid homology in the VR8 region was more profound (100%). Amino acid alignment (Fig. 3B) revealed only five changes in SC4 with respect to the prototype strain, S2. The changes were one each in VR3 and VR9 and two in VR5. One was outside the variable domain. In antigenic region A (87– 101), the alanine (A) in DS-1 was replaced by threonine (T)
35
at 87. Antigenic regions B and C were similar to DS-1. These are sites that have been shown to be involved in the neutralization of serotype G2 viruses by N-MAbs (Lazdins et al., 1995). Overall, there was a difference of 12 amino acids between the local strain SC134 and the prototype strain, ST3 (Fig. 3C). Out of this, one each was present in regions VR1, VR4 and VR6. In VR7, there were three and in VR9, there were two. No changes were there in VR2, VR3, VR5 and VR8. Four of the changes were outside the variable domains. The VP7 glycoprotein (34–38 kDa in size) is the major neutralization antigen of rotaviruses detected by hyperimmune antiserum and serves as the basis for determination of serotype (Bastardo et al., 1981; Killen and Dimmock, 1982; Lazdins et al., 1985; Sonza et al., 1984). Comparison of the VP7 of strains belonging to 14 rotavirus serotypes indicated that there are nine regions viz. VR1(aa9–20), VR2(25–32), VR3(37–53), VR4(65– 76) VR5(87– 100), VR6(119–132), VR7(141–150), VR8(208–224) and VR9(235–
Fig. 2. Phylogram of VP7 proteins deduced from local strains, WD33 (G1), SC4 (G2) and SC134 (G4). The strains, d, DS1 and ST3 were used for the human-rhesus rotavirus tetravalent vaccine formulation. The phylogenetic tree was generated from a VP7 amino acid multiple sequence file using the Prodist and Kitsch modules of the Phylip 3.5 program to calculate a distance matrix and draw the tree.
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Fig. 3. Comparison of the predicted amino acid sequence of WD33, SC4 and SC134 with that of the respective reference strains, Wa, S2 and ST3.The positions of the variable domains, VR1-VR9 are indicated.
D. Khetawat et al. / Virus Research 87 (2002) 31–40
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Table 3 List of strains used in the construction of the phylogenetic trees along with the respective accession numbers Serial No.
G1 isolates (accession no.)
G2 isolates (accession no.)
G4 isolates (accession no.)
1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30. 31. 32. 33. 34. 35. 36. 37. 38. 39. 40. 41.
brz-2 (U26362) brz-3 (U26363) ban-48 (U26364) brz-4 (U26365) ban-59 (U26366) brz-5 (U26367) brz-6 (U26368) cos-69 (U26369) cos-70 (U26370) chi-45 (U26371) chi-46 (U26372) egy-7 (U26373) egy-8 (U26374) isr-55 (U26375) isr-56 (U26376) kor-54 (U26377) kor-64 (U26378) wadc-6 (U26379) md-216 (U26380) mo-12 (U26381) mo-15 (U26382) neb-49 (U26383) ohio-11 (U26384) ohio-25 (U26385) ohio-40 (U26386) ohio-64 (U26387) ri-14 (U26388) ri-39 (U26389) ri-43 (U26390) rus-91 (U26391) tn-16 (U26392) tn-39 (U26393) va-12 (U26394) zam-52 (U26395) jpn-417 (D16328) jpn-418 (D16327) jpn-421 (D16326) Wa (K02033) d (M37340) aus-91 (M93006) WD33 (Y18786)
1992A (U73947) 1992B (U73948) 1992C (U73949) 1994A (U73950) 1994B (U73952) 1994C (U73951) 1994D (U73953) 1994E (U73954) 1995A (U73955) 1995B (U73956) 1995C (U73957) 1995D (U73958) CHIN1 (D50112) CHIN3 (D50113) CHIN5 (D50114) CHIN7 (D50115) CHIN8 (D50116) PAK458 (D50126) PAK426 (D50125) JAPAN076 (D50119) JAPAN038 (D50118) JAPAN0022 (D50117) JAPAN085 (D50120) JAPAN137 (D50121) JAPAN21 (D50122) JAPAN58 (D50123) DS1 (M37348) S2 (M11164) KUN (D50124) TMC-II (D50127) RV5 (M28377) HN126 (M37349) TW3780 (AF044348) TF85 (AF106299) TA26 (AF106284) M48 (L11605) SC4 (AJ293718)
Rota 104 (AB012065) Rota 231 (AB012066) Rota 349 (AB012067) Rota 399 (AB012068) Rota 49 (AB012069) Rota 39 (AB012070) Rota 513 (AB012071) Rota 544 (AB012072) Rota 551 (AB012073) Rota 554 (AB012074) Rota 106 (AB012075) Rota 3 (AB012076) Rota 331 (AB012077) Rota Hochi (AB012078) Rota Odelia (AB012079) GR442/86 (AF161817) GR630/86 (AF161818) NB111/86 (AF161819) NB187/86 (AF161820) NB123/86 (AF161821) GR846/86 (AF161822) GR1107/86 (AF161823) GR828/86 (AF170834) GR833/86 (AF170835) GR856/86 (AF170836) GR1106/86 (AF170837) ST3 (X13603) PV5249 (M86490) VA79 (M86834) PV5257 (M86832) VA75 (M86833) SC134 (AJ278217)
242), of the linear amino acid sequence of VP7 which are highly divergent (Green et al., 1989). Each of these regions is highly conserved in human rotavirus strains within the same serotype. There is sufficient conservation of sequence of human rotavirus strains within a serotype that it is possible to predict the serotype of an isolate by
direct sequence analysis of two of these variable regions (VR5 and VR8) of VP7 (Green et al., 1987, 1988, 1989). Immunological studies using neutralizing monoclonal antibodies (N-MAbs) directed at VP7 have been classified into three functional groups: monotype-specific, serotype-specific and cross-reactive. Studies involving generation of
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mutants resistant to neutralization by N-MAbs and sequencing of the gene encoding VP7 of such mutants indicated that the VR5 (region A, amino acids 87–96) is involved in monotype specific, serotype specific or cross-reactive neutralization and both VR7 (region B, amino acids 145– 150) and VR8 (region C, amino acids 211– 223) contain monotype-specific or serotype specific neutralization sites (Dyall-Smith et al., 1986; Mackow et al., 1988; Taniguchi et al., 1988; Matsui et al., 1989; Coulson and Kirkwood, 1991; Kobayashi et al., 1991a,b). These regions exhibit clustered amino acid variation, most notably between rotaviruses of different G type (Dyall-Smith et al., 1986; Green et al., 1987, 1988). Mutations in the A and C regions have been reported in variants of G1 viruses Ku (Taniguchi et al., 1988; Kobayashi et al., 1991a), RV-4 (Coulson and Kirkwood, 1991) and K8 (Kobayashi et al., 1991b). Mutations in these regions were also detected in variants of the G2 virus DS-1 (Dunn et al., 1993b), G3 viruses (Mackow et al., 1988; Hoshino et al., 1994), G9 viruses (Kirkwood et al., 1993) and G4, 6, 9 and 10 viruses (Hoshino et al., 1994). It seems possible that the circulating G1, G2 and G4 strains may have significant differences from ‘D’, ‘DS1’ and ‘ST3’ in their VP7 gene sequences and this, in turn, may cause significant differences in anti-D, DS1 and ST3 neutralizing antibody titers found in vaccinees relative to the titers of the circulating strains examined. From our study, it is apparent that G1, G2 and G4 genotypes of rotavirus are, at present, prevalent in this part of the country. The phylogenetic analysis of representative strains demonstrates the relation with the genotypes incorporated in the rhesus-rotavirus tetravalent vaccine formulation. The first human rotavirus vaccine (a human-animal tetravalent reassortant virus mixture) was licensed in the US in 1998 (Parashar et al., 1998). The human-rhesus rotavirus reassortant vaccines were developed with the goal of combining the specificity of the epidemiologically important VP7 serotypes with the attenuation phenotype of RRV, MMU18006 (Midthun et al., 1985, 1986). Human-RRV reassortant strains were recovered after coinfection of AGMK cell cultures with the RRV vaccine strain, MMU18006 and human ro-
tavirus strain D, DS-1 or ST3. The D (VP7 serotype 1) and DS-1 (VP7 serotype 2) strains were initially detected in stools of children hospitalized with diarrhea and were then passaged in gnotobiotic calves, whereas, ST3 (VP7 serotype 4) was derived from the stool of an asymptomatic new born infant and was isolated in AGMK cells. Vaccine lots were subsequently prepared in DBSFRhL2 cells, a semi-continuous diploid cell strain developed as a suitable vaccine substrate. The efficacy of this vaccine in developing countries has been variable, but, in a recent trial involving more than 2200 underprivileged urban children in Venezuela, vaccine efficacy approached levels seen in industrialized countries (48% against all episodes of diarrhea, 70% against episodes requiring hospitalization, 75% against dehydrating illness and 88% against severe episodes of rotavirus diarrhea) (Perez-Schael et al., 1997). A better understanding of the population genetics of rotavirus will assist in the development of more effective vaccine formulations and strategies. The information on rotavirus genotypes/serotypes seems to play an important role in inducing immunity and subsequent vaccine development (Jin et al., 1996; Gentsch et al., 1996; Hoshino and Kapikian, 2000). Variation within a genotype is well documented and subtypes within genotype has also been described (Palombo et al., 1993). Strains of less common or unusual serotypes, which also cause diarrhea have been described and constitute 14% of global isolates. In particular, recent surveys carried out in Brazil, India and Bangladesh, demonstrated the emergence of serotypes G5 and G9 that are not covered by the RRV-TV vaccine (Kapikian and Chanock, 1996). Again, difference in antigenic responses has also been reported, based on the genetic variation (Coulson et al., 1996; Diwakarla and Palombo, 1999). In particular, variation is seen in VP7, the viral protein targeted by current vaccine strategies. Ultimately, the effect that diversity of the viral population has on the success of human rotavirus vaccines and the role that mass vaccination will have on rotavirus evolution, should be determined from surveillance and epidemiological studies of clinical isolates before and after the introduction of vaccination programs.
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Acknowledgements We thank Dr Jon R. Gentsch, Viral Gastroenteritis Section, Center for Disease Control and Prevention, Atlanta, GA, for his advice and critical comments. The sequences of the VP7 genes used in this study were submitted to the Genbank and the assigned Accession Nos. are Y18786 (WD33), AJ293718 (SC4) and AJ278217 (SC134).
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