JOURNAL OF CLINICAL MICROBIOLOGY, JUlY 1994, p. 1820-1822
Copyright X 1994, American Society for Microbiology
Vol. 32, No. 7
Characterization of Rotavirus Strains from Newborns in
New Delhi, India
BIMAL K. DAS,' JON R. GENTSCH,2* HELEN G. CICIRELLO,2 PATRICIA A. WOODS,2 AARTI GUPTA,'
MADHUMATI RAMACHANDRAN,' RAMESH KUMAR,1 M. K. BHAN,' AND ROGER I. GLASS2
Department ofPaediatrics and Microbiology, Division of Gastroenterology and Enteric Infections, All India Institute of
Medical Sciences, Ansari Nagar, New Delhi 11029, India, 1 and The Viral Gastroenteritis Section,
Division of Viral and Rickettsial Diseases, National Centerfor Infectious Diseases,
Centers for Disease Control and Prevention, Atlanta, Georgia 303332
Received 8 December 1993/Returned for modification 18 April 1994/Accepted 18 April 1994
Between 1986 and 1993, 72% of rotavirus strains isolated from newborns at five hospitals in New Delhi,
India, had long electropherotypes, subgroup II VP6 antigens, and G and P genotypes (G9P11) identical to those
ofprototype strain 116E. A novel strain with aGAP6genotype, representing 13% of the isolates, was identified.
These results demonstrate that GgPll andGAP6rotavirus strains are common in nurseries in New Delhi.
Serologic characterization of rotavirus strains indicates that
outer capsid proteins VP4 and VP7 independently induce
type-specific neutralizing antibodies which have been used to
classify rotaviruses into G (VP7) and P (VP4) serotypes (11).
Among rotavirus strains isolated from children with diarrhea,
four major G serotypes, G1 to G4, have been shown to be
epidemiologically important by enzyme-linked immunosorbent
assay with VP7-specific monoclonal antibodies, but until re-
cently, analogous methods to study the P serotypes ofcommon
human rotavirus strains (i.e., G serotypes 1 to 4) were not
available (16, 17). As a result, nucleic acid-based (genotyping)
methods that detect genetically distinct VP4 genes and accu-
rately predict P serotypes have been developed (8). VP4 genes
that are distinct at the level of nucleotide and deduced amino
acid sequences have been referred to as P types (or P
genotypes) (6). Although a nomenclature for corresponding P
serotypes has not been agreed upon, some investigators use the
same numbering system for genotypes and serotypes and that
is the convention used in this report (6, 14). The largest survey
of rotavirus field isolates indicated that genotypes P8 (strain
Wa-like VP4 gene) and P4 (strain RV5-like VP4 gene) are by
far the most common among more than 400 strains from
several areas of the world (15), while the largest survey in
which both G and P genotypes were determined for the same
strain indicated that G1P8 (strain Wa-like VP7 and VP4 genes)
is the most common in Malaysia (13).
We recently demonstrated that asymptomatic neonatal ro-
tavirus infections of children in New Delhi, India, reduced the
frequency of subsequent cases of severe rotavirus diarrhea by
46%, essentially confirming the work of Bishop and coworkers
and raising the possibility that strains related to New Delhi
isolate 116E may be effective as vaccines (1, 2). Previously
isolated neonatal rotaviruses belonged to serotypes G,P6
(strain M37), G2P6 (strain 1076), G3P6 (strain McN13), and
G4P6 (strain ST3) (9, 12). We subsequently showed by sero-
logic and sequence analyses that prototype strain 116E belongs
to serotype Gg and genotype Pl and has not been previously
isolated from humans (4). Since the prototype strains for
serotypes Gg (human isolate W161, serotype G9P8) andPl,
(bovine isolate B223, serotype G1oP1 ) have P and G types,
*Corresponding author. Phone: (404) 639-3577. Fax: (404) 639-
respectively, that are different from those of 116E, it was
suggested that the latter strain may be a reassortant (7).
We have recently extended these results and shown that
strains related to prototype 116E are present in five of six New
Delhi hospitals (3). In this report, we describe the complete
characterization of rotavirus strains isolated from newborns at
six government hospitals in New Delhi between 1986 and 1988
and 1992 and 1993, as well as isolates previously collected in a
longitudinal study by using G and P genotyping by reverse
analysis, and nucleotide sequencing.
To screen for genotype P11 rotavirus strains, a specific
primer, ND2, that is complementary to nucleotides (nt) 116 to
133 of the strain 116E VP4 gene was synthesized and added to
a one-amplification RT-PCR system that detects strains with
M1 2 345 678M
4 5 6 7 8
FIG. 1. RT-PCR typing of rotavirus strains. Rotavirus double-
stranded RNA was extracted from cell lysates or fecal specimens, and
5 ,ul of the eluate was analyzed (8). (A) P typing. Lanes: M, markers
(123-bp ladder; Gibco BRL, Long Island, N.Y.; marker molecular sizes
are indicated on the left in base pairs); 1 to 5, products amplified from
double-stranded RNA from human rotavirus strains possessing P types
8 (lane 1, strain Wa), 4 (lane 2, strain DS-1), 6 (lane 3, strain M37), 9
(lane 4, strain K8), 10 (lane 5, strain M37), and 11 (lane 6, strain 116E);
7 to 8, products from double-stranded RNA extracted from culture-
adapted strains (lane 7, strain 113E; lane 8, strain 218D). (B) G typing.
Lanes: M, markers (123-bp ladder); 1 to 3, products amplified from
double-stranded RNA of human rotavirus G serotype 9 (lane 1, strain
116E; lane 2, strain F45; lane 3, strain WI61); 4 to 8, products
amplified from field isolates from Costa Rica possessing serotypeGIto
G4 specificities as determined by enzyme-linked immunosorbent assay
serotyping with monoclonal antibodies.
VOL. 32, 1994
TABLE 1. Electropherotypes, G and P genotypes, and subgroups" of rotaviruses isolated from newborns in New Delhi
No. of strains with the following G genotype, P genotype, and subgroup"
G PS,GP,G, GP,G,9PS,96G,3,S1T
"Subgroups of rotavirus antigen-positive fecal specimens, as determined by the Rotaclone test (Cambridge Biosciences, Cambridge, Mass.). were determined as
described previously, with antibodies provided by H. Greenberg (10).
"Of the 67 strains tested, 64 had similar long electropherotypes and the lone SG, strain had a short electropherotype.
SG.., indicates that the strain could not be subgrouped.
dDT, dual PCR type, i.e., presence of two DNA products, consistent with the presence of two different genotypes. P6P, , dual types were verified by restriction
endonuclease analysis of both products (data not shown).
"One strain from hospital A was non-G-nongenotypeable, while the P-typing reaction for one isolate from hospital E was omitted.
1'Culture-adapted strains; all of the other strains tested were isolated directly from fecal specimens.
genotypes P4 (e.g., strain DS-1 like), P6, (e.g., strain M37 like),
P. (e.g., strain Wa like), P9 (e.g., strain K8 like), andP1,( (e.g.,
strain 69M like). The procedures for RNA extraction and
amplification were identical to those described previously,
except that 40 PCR cycles were used and the extension time
was 3 min at 720C (8).
For G genotyping (detection of VP7 genes by RT-PCR), a
consensusprimer,9 con 1, whosesequence is conservedamong
the VP7 genes of serotypes GI to G4 and G9 (GenBank
accession numbers, K02033, M11164, U04350, M21650, and
L14072) and five genotype-specific primers complementary to
variable regions of the VP7 genes of the same serotypes were
synthesized and used in an RT-PCR system analogous to that
used for P genotyping. The nucleotide positions, strain and
serotype specificities, polarities (plusor minus), andsequences
(from 5' to 3') oftype-specific complementary primers 9TI-1,
9T1-2, 9T-3P, 9T-4, and 9T-9B are as follows: 9 Con 1, nt 37 to
56, Wa, plus sense, and TAGCTCCTTTT7AATGTATGG;
9TI-1, nt 176 to 195, Wa, G,,minus sense, and TCTTGT
CAAAGCAAATAATG; 9TI-2, nt 262 to 281, S2, G2, minus
sense, and GTFlAGAAATGAYTTCTCCACT; 9T-3P, nt 484 to
503, 107EIB, G3, minus sense, and GTCCAGTTIGCAGTGT
TAGC; 9T-4, nt 423 to 440, ST3, G4, minus sense, and
GGGTCGATGGAAAATTCT; 9T-9B, nt 131 to 147, 116E,
G9, minus sense, and TATAAAGTCCATFGCAC. The ex-
pected molecular sizes of the RT-PCR products of the primer-
pair consisting of 9 con 1 and 9T1-1, 9TI-2, 9T-3P, 9T-4, or
9T-9B were 158, 244, 466, 403, or 110 bp, respectively. The
strain specificities of the P1Irand G9-specific RT-PCR primer
pairs are shown in Fig. IA and B.
A summary of the characteristics of 75 strains from new-
borns is presented in Table 1. The type designations of the
strains identified here by genotyping conform to the sugges-
tions of Estes and Cohen (6). G and/or Pserotype designations
have onlybeengiven to strains analyzed bycross-neutralization
tests. Nucleotide sequence analysis of variable regions of the
VP4 and VP7 genes of two strains from newborns demon-
strated that our RT-PCR typing method accurately predicted
their G and P genotypes (7).
Recently, we reported the isolation of novel rotavirus strains
from newborns with serotype G9 and genotype PI1 specificity
(4). These findings have since been extended to five other
hospitals in New Delhi where strains related to prototype
strain 1 16E were detected (3). In this report, we more com-
pletely characterized all of the strains isolated in the 1993
study, as well as strains isolated from two of the hospital
nurseries between 1986 and 1992. Our results strongly suggest
that genotype PI11strains are common in New Delhi. A second
novel strain from newborns-genotype G9P,-identified
two of the hospitals completely replaced the genotype G9PII
strains found in hospital A between 1992 and 1993. Although
the genotype P6 VP4gene of these strains is related to those of
previously identified strains from newborns (e.g., Venezuelan
strain M37, serotype G1P6), genotype G9P6strains have not
been isolated before (9). It is possible that these strains arose
by reassortment, since we found that isolates with dual types
(typesP6andPI11andG1Iand G9) were isolated from the same
infants in hospital B in 1992, and we subsequently identified
genotypes G9P6 andG9P11I cocirculating in 1993 at hospital B.
Regardless of their origin, the isolation of three different,
unique rotaviruses from newborns in New Delhi and Banga-
lore suggests that additional surveillance should be conducted
to determine the prevalent strains from newborns in other
areas of India and to investigate if any of these novel rotavi-
ruses are common in children with diarrhea (3, 5). These
studies will be important to assess the possible utility of such
strains as vaccine candidates.
We thank Ann Mather for help in editing the manuscript, Brian
Holloway of the CDC Molecular Biology Core Facility for primer
synthesis, and Taka Hoshino for providing several rotavirus strains.
This study was supported in part by a grant from the INDO-U.S.
Vaccine Action Program.
1. Bhan, M. K., J. F. Lew, S. Sazawal, B. K. Das, J. R. Gentsch, and
infection against subsequent diarrhea. J. Infect. Dis. 168:282-287.
2. Bishop, R. F., G. L. Barnes, E. Cipriani, and J. S. Lund. 1983.
Clinical immunity after neonatal rotavirus infection: aprospective
longitudinal study in young children. N. Engl. J. Med. 309:72-76.
3. Cicirello, H., B. K. Das, A. Gupta, M. K. Bhan, J. R. Gentsch, R.
Kumar, and R. I. Glass. High prevalence of rotavirus infection
among neonates born at hospitals in Delhi, India: predisposition
of newborns for infection with unusual rotavirus strains. Pediatr.
Infect. Dis. J., in press.
4. Das, B. K., J. R. Gentsch, Y. Hoshino, S.-I. Ishida, 0. Nakagomi,
M. K. Bhan, R. Kumar, and R. I. Glass. 1993. Characterization of
I. Glass. 1993. Protection conferred by neonatal rotavirus
J. CLIN. MICROBIOL. Download full-text
the G serotype and genogroup of New Delhi newborn rotavirus
strain 116E. Virology 197:99-107.
5. Das, M., S. J. Dunn, G. N. Woode, H. B. Greenberg, and C. D. Rao.
1993. Both surface proteins (VP4 and VP7) of an asymptomatic
neonatal rotavirus strain (1321) have high levels of sequence
identity with the homologous proteins of a serotype 10 bovine
rotavirus. Virology 194:374-379.
6. Estes, M. K., and J. Cohen. 1989. Rotavirus gene structure and
function. Microbiol. Rev. 53:410-449.
7. Gentsch, J., B. K. Das, B. Jiang, M. K. Bhan, and R. I. Glass. 1993.
Similarity of the VP4 protein of human rotavirus strain 116E to
that of the bovine B223 strain. Virology 194:424-430.
8. 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.
9. Gorziglia, M., G. Larralde, A. Z. Kapikian, and R. M. Chanock.
1990. Antigenic relationships among human rotaviruses as deter-
mined by outer capsid protein VP4. Proc. Natl. Acad. Sci. USA
10. Greenberg, H., V. McAuliffe, J. Valdesuso, R. Wyatt, J. Flores, A.
Kalica, Y. Hoshino, and N. Singh. 1983. Serological analysis of the
subgroup protein of rotavirus using monoclonal antibodies. Infect.
11. Hoshino, Y., M. M. Sereno, K. Midthun, J. Flores, A. Z. Kapikian,
and R. M. Chanock. 1985. Independent segregation of two anti-
genic specificities (VP3 and VP7) involved in neutralization of
rotavirus infectivity. Proc. Natl. Acad. Sci. USA 82:8701-8704.
12. Hoshino, Y., R. G. Wyatt, J. Flores, K. Midthun, and A. Z.
Kapikian. 1985. Serotypic characterization of rotaviruses derived
from asymptomatic human neonatal infections. J. Clin. Microbiol.
13. Rasool, N. B. G., G. Larralde, and M. I. Gorziglia. 1993. Deter-
mination of human rotavirus VP4 using serotype-specific cDNA
probes. Arch. Virol. 133:275-282.
14. Snodgrass, D. R., Y. Hoshino, T. A. Fitzgerald, M. Smith, G. F.
Browning, and M. Gorziglia. 1992. Identification of four VP4
serological types (P serotypes) of bovine rotavirus using viral
reassortants. J. Gen. Virol. 73:2319-2325.
15. Steele, A., D. Garcia, J. Sears, G. Gerna, 0. Nakagomi, and J.
Flores. 1993. Distribution of VP4 gene alleles in human rotavi-
ruses by using probes to the hyperdivergent region of the VP4
gene. J. Clin. Microbiol. 31:1735-1740.
16. Taniguchi, K., T. Urasawa, Y. Morita, H. B. Greenberg, and S.
Urasawa. 1987. Direct serotyping of human rotavirus in stools
using serotype 1-, 2-, 3-, and 4-specific monoclonal antibodies to
VP7. J. Infect. Dis. 155:1159-1166.
17. Woods, P. A., J. Gentsch, V. Gouvea, L. Mata, A. Simhon, M.
Santosham, Z.-S. Bai, S. Urasawa, and R. I. Glass. 1992. Distri-
bution of serotypes of human rotavirus in different populations. J.
Clin. Microbiol. 30:781-785.