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The VP8(star) Domain of Neonatal Rotavirus Strain G10P[11] Binds to Type II Precursor Glycans

American Society for Microbiology
Journal of Virology
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Naturally occurring bovine-human reassortant rotaviruses with a P [11] VP4 genotype exhibit a tropism for neonates. Interaction of the VP8* domain of the spike protein VP4 with sialic acid was thought to be the key mediator for rotavirus infectivity. However, recent studies indicate a role for non-sialylated glycoconjugates including histo-blood group antigens (HBGAs) in the infectivity of human rotaviruses. We sought to determine if the bovine rotavirus-derived VP8* of a reassortant neonatal G10P [11] virus interacts with hitherto uncharacterized glycans. In an array screen of >600 glycans, VP8* P [11] showed specific binding to glycans with the Galβ1-4GlcNAc motif, which forms the core structure of type II glycans and is the precursor of H type II HBGA. The specificity of glycan binding was confirmed through hemagglutination assays; GST-VP8* P [11] hemagglutinates type O, A and B red blood cells as well as pooled umbilical cord blood erythrocytes. Further, G10P [11] infectivity was significantly enhanced by the expression of H type II HBGA in CHO cells. The bovine origin VP4 was confirmed as essential for this increased infectivity using laboratory-derived reassortant viruses generated from sialic acid binding rotavirus SA11-4F and a bovine G10P [11] rotavirus B223. The binding to a core glycan unit has not been reported for any rotavirus VP4. Core glycan synthesis is constitutive in most cell types and modification of these glycans is thought to be developmentally regulated. These studies provide the first molecular basis for understanding neonatal rotavirus infections, indicating that glycan modification during neonatal development may mediate age-restricted infectivity of neonatal viruses.
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Published Ahead of Print 24 April 2013.
2013, 87(13):7255. DOI: 10.1128/JVI.03518-12. J. Virol.
Prasad and Mary K. Estes
Robert F. Ramig, Jacques Le Pendu, B. V. Venkataram
E. Crawford, Rita Czako, David F. Smith, Gagandeep Kang,
Sasirekha Ramani, Nicolas W. Cortes-Penfield, Liya Hu, Sue
Glycans
Strain G10P[11] Binds to Type II Precursor
The VP8* Domain of Neonatal Rotavirus
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The VP8* Domain of Neonatal Rotavirus Strain G10P[11] Binds to
Type II Precursor Glycans
Sasirekha Ramani,
a
Nicolas W. Cortes-Penfield,
a
Liya Hu,
b
Sue E. Crawford,
a
Rita Czako,
a
David F. Smith,
c
Gagandeep Kang,
d
Robert F. Ramig,
a
Jacques Le Pendu,
e
B. V. Venkataram Prasad,
a,b
Mary K. Estes
a
Departments of Molecular Virology and Microbiology
a
and Verna and Marrs McLean Department of Biochemistry and Molecular Biology, Baylor College of Medicine,
Houston, Texas, USA
b
; Department of Biochemistry, Emory University School of Medicine, Atlanta, Georgia, USA
c
; Christian Medical College, Vellore, Tamilnadu, India
d
;
INSERM, U892, and CNRS, UMR 6299, Université de Nantes, Nantes, France
e
Naturally occurring bovine-human reassortant rotaviruses with a P[11] VP4 genotype exhibit a tropism for neonates. In-
teraction of the VP8* domain of the spike protein VP4 with sialic acid was thought to be the key mediator for rotavirus
infectivity. However, recent studies have indicated a role for nonsialylated glycoconjugates, including histo-blood group
antigens (HBGAs), in the infectivity of human rotaviruses. We sought to determine if the bovine rotavirus-derived VP8* of
a reassortant neonatal G10P[11] virus interacts with hitherto uncharacterized glycans. In an array screen of >600 glycans,
VP8* P[11] showed specific binding to glycans with the Gal1-4GlcNAc motif, which forms the core structure of type II
glycans and is the precursor of H type II HBGA. The specificity of glycan binding was confirmed through hemagglutination
assays; GST-VP8* P[11] hemagglutinates type O, A, and B red blood cells as well as pooled umbilical cord blood erythro-
cytes. Further, G10P[11] infectivity was significantly enhanced by the expression of H type II HBGA in CHO cells. The bo-
vine-origin VP4 was confirmed to be essential for this increased infectivity, using laboratory-derived reassortant viruses
generated from sialic acid binding rotavirus SA11-4F and a bovine G10P[11] rotavirus, B223. The binding to a core glycan
unit has not been reported for any rotavirus VP4. Core glycan synthesis is constitutive in most cell types, and modification
of these glycans is thought to be developmentally regulated. These studies provide the first molecular basis for understand-
ing neonatal rotavirus infections, indicating that glycan modification during neonatal development may mediate the age-
restricted infectivity of neonatal viruses.
Interaction with cell surface glycans is a critical step in the initi-
ation of many enteric infections (1). For rotavirus, this is medi-
ated by the outer capsid spike protein VP4 through its glycan
binding domain, VP8*. Sialic acid (Sia) is a key binding partner for
VP8*; previous studies have shown that some animal rotaviruses
bind to terminal Sia residues on cell surfaces and some human
rotaviruses interact with gangliosides, such as GM1, that contain
internal Sia moieties (26). Recently, some human rotaviruses
have been shown to interact with nonsialylated glycoconjugates,
including histo-blood group antigens (HBGAs) that are found in
mucosal secretions and on the surface of epithelial cells (79).
Structural studies on the VP8* of one such human rotavirus,
HAL1166, showed that HBGA binding occurs in the same pocket
where Sia binds in animal rotaviruses and that binding is mediated
by specific sequence changes in this region. Further, the infectivity
of HAL1166 is enhanced by HBGA expression in vitro (7). These
new data are shifting the paradigm in understanding human ro-
tavirus infectivity, and while they may explain zoonotic transmis-
sion of some animal rotaviruses, their relevance to different hu-
man strains remains unclear.
Human rotaviruses are widely known to be the leading cause of
diarrheal mortality in children worldwide. However, rotavirus in-
fections in neonates are distinct from those in older children. Neo-
natal infections are predominantly asymptomatic and are often
associated with unusual rotavirus strains. These strains appear to
be geographically restricted, show remarkable stability, and can
persist in specific settings for long periods of time (10,11). Rota-
viruses are classified into G and P genotypes using a binary no-
menclature system based on the sequence variability in the genes
encoding the outer capsid glycoprotein VP7 (G type) and pro-
tease-sensitive spike protein VP4 (P type) (12). A group of un-
usual rotaviruses possessing the P[11] VP4 type and a G9 or G10
VP7 genotype has been associated with neonatal infections in In-
dia. Of these, the G10P[11] rotaviruses were initially detected in
diarrheal samples from cattle in many studies and in some asymp-
tomatic infections in neonates (1315). The understanding of the
epidemiology of these viruses, however, changed when a 4-year
hospital surveillance study involving 1,300 neonates in southern
India showed that nearly 50% of neonates were positive for rota-
virus and 81% of the samples were genotyped as G10P[11]; fur-
thermore, the virus was significantly associated with gastrointes-
tinal symptoms in this study (16). In concomitant hospital-based
surveillance studies for rotavirus diarrhea among 960 infants from
1 month to 36 months of age, only 1 case of G10P[11] was seen
out of 342 rotavirus-positive cases, clearly indicating a predilec-
tion of this strain for neonates (17). A high incidence of neonatal
infections with the G10P[11] strain was also detected in a com-
munity-based birth cohort in this region, confirming the predi-
lection of this strain for neonates (18,19). Through whole-ge-
Received 21 December 2012 Accepted 12 April 2013
Published ahead of print 24 April 2013
Address correspondence to Mary K. Estes, mestes@bcm.edu.
S.R. and N.W.C.-P. contributed equally to this article.
Supplemental material for this article may be found at http://dx.doi.org/10.1128
/JVI.03518-12.
Copyright © 2013, American Society for Microbiology. All Rights Reserved.
doi:10.1128/JVI.03518-12
July 2013 Volume 87 Number 13 Journal of Virology p. 7255–7264 jvi.asm.org 7255
on June 13, 2014 by guesthttp://jvi.asm.org/Downloaded from
nome sequencing of a clinical isolate, the neonatal G10P[11] virus
was identified to be a bovine-human reassortant, with the genes
encoding nonstructural proteins NSP1 to NSP3 being of human
rotavirus origin and the remaining 8 genes, including VP4, being
of bovine rotavirus origin (20). Another P[11] rotavirus strain
associated with neonatal infections is G9P[11]. This strain causes
asymptomatic neonatal infections in northern India and forms
the basis for a new rotavirus vaccine currently in phase III trials
(21,22). G9P[11] is also a bovine-human reassortant virus, with
all genes being of human rotavirus origin except VP4, which is of
bovine rotavirus origin (23,24).
Given the high disease burden of rotavirus gastroenteritis and
the cocirculation of many genotypes, including globally prevalent
strains, the predominance of a single genotype in this age-re-
stricted population sharply contrasts with the strain diversity seen
among older children (16,17,25). With recent evidence for the
interaction of the VP8* of human rotaviruses with novel glycan
partners, we sought to determine if the bovine rotavirus-derived
VP8* of neonatal G10P[11] rotavirus interacts with hitherto un-
characterized glycan partners and whether this interaction is sig-
nificant for infection.
MATERIALS AND METHODS
Comparison of VP8* sequences. To determine if the VP8* amino acid
sequence of P[11] rotaviruses was different from that of other strains with
known glycan partners, multiple alignments were performed using the
Clustal W algorithm on BioEdit software (version 7.0.5.3). The G10P[11]
sequences included those from (i) a human neonatal isolate (N155) whose
whole genome has previously been sequenced (20), (ii) a human neonatal
isolate (N1509) that has been adapted to culture through multiple pas-
sages on African green monkey kidney epithelial (MA104) cells, and (iii) a
bovine G10P[11] isolate (strain B223). Both N155 and N1509 were iso-
lated from symptomatic neonates with feed intolerance. The VP8* amino
acid sequences of P[11] rotaviruses were compared to the VP8* amino
acid sequences of other animal and human rotaviruses with known glycan
partners (Fig. 1). These included sialidase-sensitive animal rotavirus
strains that bind to glycans with a terminal Sia moiety (rhesus rotaviruses
RRV and SA11, porcine rotavirus CRW-8) and human rotaviruses that
bind to glycans with internal Sia residues (Wa and DS-1). Wa and DS-1
represent the two most common global genotypes, G1P[8] and G2P[4],
respectively. In addition, the VP8* amino acid sequence of a P[14] siali-
dase-insensitive human rotavirus, HAL1166, that binds to A-type HBGA
at the same location as Sia in the VP8* of animal rotaviruses was also
included in the analysis.
GST-VP8* protein expression and purification. The gene sequences
of VP8* corresponding to amino acids (aa) 64 to 224 of human neonatal
G10P[11] rotaviruses N155 and N1506 as well as the bovine G10P[11]
rotavirus B223 were synthesized (Epoch Life Science) and cloned with an
N-terminal glutathione S-transferase (GST) tag into a pGEX-2T expres-
sion vector (GE Healthcare) (7). The recombinant proteins were ex-
pressed in Escherichia coli BL21(DE3) cells (Novagen) and purified using
glutathione Sepharose 4 Fast Flow chromatography medium (GE Health-
care). The VP8* segments of SA11 variant 4F (SA11-4F) and human ro-
tavirus Wa were also expressed and purified for use as controls. The pu-
rified GST-VP8* proteins were used in glycan array and hemagglutination
assays.
Glycan array screen for GST-VP8* proteins. The carbohydrate bind-
ing specificity of GST-VP8* P[11] was determined using a glycan array
comprised of 600 glycans (v5.0; Consortium for Functional Glycomics,
Protein-Glycan Interaction Core-H, Emory University School of Medi-
cine [http://www.functionalglycomics]) as previously described (7).
Briefly, the recombinant protein from neonatal G10P[11] N155 was used
in decreasing concentrations (200, 20, 2.0, and 0.2 g/ml) in individual
arrays and detected using a fluorescently labeled anti-GST monoclonal
antibody (Sigma). The strength of binding to a glycan was expressed in
terms of relative fluorescence units (RFU). To normalize the results be-
tween the different arrays, a rank was assigned to each glycan using the
formula 100 (test glycan RFU/highest number of RFU in that assay). An
average of the ranks determined the final rank for each glycan (26). Sim-
ilar glycan array studies were carried out with GST-VP8* proteins from
SA11-4F, Wa, N1509, and B223. These proteins were tested in a single
concentration (200 g/ml) on the array.
Hemagglutination assay. Adult human red blood cells (RBCs) corre-
sponding to blood types O, A1, A2, and B were obtained from Immucor
Inc. Pooled umbilical cord blood erythrocytes were obtained from My-
biosource. The cells were centrifuged for 10 min at 500 g, and 0.5%
suspensions of each RBC type were prepared in 0.85% saline (pH 6.2). The
GST-VP8* proteins were serially diluted on 96-well V-bottom plates
(Nunc) and mixed with an equal volume of each RBC suspension. The
suspension was incubated for about1hat4°Candassessed for hemag-
glutination. The hemagglutination titer was recorded as the highest dilu-
tion of sample that resulted in complete hemagglutination. Recombinant
Norwalk virus virus-like particles (VLPs) known to hemagglutinate type
A and type O but not type B RBCs and GST-VP8* P[14] from rotavirus
strain HAL1166, which hemagglutinates type A RBCs, were included as
positive controls. The assay was also performed with GST to rule out false
positivity due to the GST tag.
To determine the effect of neuraminidase treatment of RBCs on hem-
agglutination, O-type RBCs were treated with neuraminidase from Vibrio
cholerae (Sigma). Briefly, a 10% suspension was prepared using 10 lof
packed RBCs in 250 mM sodium acetate buffer (pH 5.8) containing bo-
vine serum albumin (1.25 mg/ml), NaCl (5 mM), and CaCl
2
(4 mM). The
RBCs were treated with or without 25 mU neuraminidase for2hat37°C
(modified from reference 27). Following incubation, the cells were
washed and hemagglutination assays were carried out as described above.
The neuraminidase treatment studies were carried out with a subset of the
GST-VP8* proteins. These included VP8* from human and bovine
G10P[11] rotaviruses (N155 and B223, respectively), SA11-4F, and
HAL1166.
Cell lines and virus strains for binding and infectivity assays. Bind-
ing and infectivity assays were performed on Chinese hamster ovary
(CHO) cells that differ in the production of HBGAs (7). The parental CHO
cells do not express HBGAs (H/A/B). Single-transfectant CHO cells
that stably express the enzyme fucosyltransferase (Fut2) express the H antigen
(H/A/B). Double-transfectant CHO cells expressing Fut2 and either
A-type glycosyltransferase or B-type glycosyltransferase express the A antigen
(H/A/B) or B antigen (H/A/B), respectively (28). Polylac-
tosamines were assayed on CHO cells by flow cytometry using the biotin-
ylated lectins STL from Solanum tuberculosum (potato; Biovalley), LEL
from Lycospersum (tomato; Biovalley), and streptavidin-peridinin chlo-
rophyll protein conjugate (BD Biosciences) at 1 g/ml.
The binding studies were carried out using VP8* P[11] from N155 and
VP8* P[14]. Infectivity on CHO cells was assessed using a panel of virus
strains. These included the human neonatal G10P[11] rotavirus isolate
(N1509) that was adapted to culture through multiple passages on MA104
cells, well-characterized laboratory strains (simian rotavirus SA11 variant
4F [SA11-4F], human rotaviruses Wa and HAL1166, and bovine
G10P[11] rotavirus B223), as well as 6 reassortant viruses (R-179, R-144,
R-141, R-491, R-004, and R-198) generated using SA11-4F and B223 as
parental strains. The reassortant viruses differed in their overall genetic
background (see Fig. 6), and their characterization has been described
previously (29,30).
Binding and infectivity assays on CHO cells. Binding of VP8* to the
different CHO cells was assessed by flow cytometry using fluorescein iso-
thiocyanate (FITC)-labeled GST-VP8* proteins. The binding studies were
carried out on parental CHO cells or CHO cells stably expressing the
H-type or the A-type glycosyltransferase with FITC-GST-VP8* P[11]
from human neonatal G10P[11] N155. FITC-GST and FITC-GST-V8*
Ramani et al.
7256 jvi.asm.org Journal of Virology
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P[14] from HAL1166 were included as controls. Briefly, 2 mg/ml of pu-
rified recombinant GST, GST-VP8* P[11], or P[14] in phosphate-buff-
ered saline (pH 8.0) was labeled using an FITC antibody-labeling kit
(Thermo Scientific). The proteins were incubated with one vial of FITC
reagent each at room temperature for 1 h with protection from light. The
labeled proteins were then purified using the purification resin provided
in the kit and stored at 4°C before examining their binding to CHO cells.
The binding studies were carried out at 4°C for 30 min using 25 gof
protein for 1 10
6
to 2 10
6
cells. In addition, binding studies were also
carried out following treatment of CHO cells with neuraminidase at 37°C
for1hataconcentration of 50 mU per 10
6
cells.
Virus titers on the parental and the singly and doubly transfected CHO
cell lines were determined using fluorescent focus assays (3). Briefly, the
cells were grown to confluence on 96-well plates (Costar; Corning) and
inoculated with 2-fold dilutions of trypsin-activated virus. After 12 to 14 h
of infection, the cells were fixed with ice-cold methanol and stained with a
rabbit polyclonal antirotavirus antibody and a fluorescently labeled don-
key anti-rabbit IgG secondary antibody (Invitrogen). Virus titers were
calculated from dilutions that gave countable numbers of foci (20 to 200)
per well. Differences in virus titers between cells lines were compared
using a two-tailed Student ttest.
Nucleotide sequence accession number. The VP8* sequence of neo-
natal G10P[11] N1509 was deposited in GenBank and assigned the acces-
sion number KC807203.
RESULTS
The VP8* sequence of P[11] rotaviruses is distinct in the glycan
binding domain. Many animal rotaviruses are known to bind
glycans with a terminal Sia residue on cell surface glycans. The
crystal structures of the VP8* of animal rotaviruses RRV and
CRW-8 with bound Sia have been determined, and the residues
FIG 1 The VP8* P[11] sequence is distinct from the sequence in rotavirus strains with known glycan partners. Alignment of the VP8* amino acid sequences from
neonatal G10P[11] rotavirus isolates (N155 [GI:164632843] and culture-adapted neonatal isolate N1509) with the VP8* amino acid sequences from a bovine
(Bo) G10P[11] isolate (B223; GI:408959), simian (Si) rotaviruses RRV (GI:61869) and SA11 (GI:61892), SA11 variant 4F (GI:61946), porcine (Po) rotavirus
CRW-8 (GI:226699783), and human (Hu) rotavirus strains Wa (GI:333781), DS-1 (GI:28268531), and HAL1166 (GI: 452131) is shown. The species of origin,
the names of the isolates, and their VP4 types are indicated. *, residues known to interact with Sia for the animal viruses RRV, SA11, and CRW-8. The VP8* amino
acid sequence of cell culture-adapted human neonatal rotavirus strain N1509 G10P[11] was 98% identical to that of clinical isolate N155. N155 and N1509
showed the same amino acid changes in the Sia or HBGA binding pocket as RRV, CRW-8, and HAL1166.
Neonatal Rotavirus VP8* Binds Type II Precursor
July 2013 Volume 87 Number 13 jvi.asm.org 7257
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that interact with Sia have been identified (2,4). Studies on a
human rotavirus (HAL1166) have shown that VP8* binds A-type
HBGA in the same location as Sia in the VP8* of the animal viruses
(7). An insertion in amino acid position 187 of HAL1166 VP8*
results in a change in the side chain orientation of the conserved
residues R101 and Y188, which sterically hinders Sia binding in
this pocket but allows A-type HBGA binding (7). The VP8* do-
mains of neonatal P[11] strains (N155 and N1509) show signifi-
cant alterations in the residues that interact with either Sia in the
VP8* domain of animal rotaviruses or the A-type HBGA in the
VP8* domain of HAL1166 (Fig. 1). In P[11] rotaviruses, R101 is
replaced by a phenylalanine, while the conserved Y188 and Y189
residues are not seen. The VP8* crystal structures of two other
human rotavirus strains representing the globally prevalent P[8]
(Wa) and P[4] (DS-1) genotypes have also been reported previ-
ously (2,5,6). Using nuclear magnetic resonance, the Wa VP8*
was shown to bind gangliosides, such as GM1, with internal Sia
residues. Although the structural determinants of binding to the
internal Sia residues are not characterized for these human vi-
ruses, the VP8* sequence of P[11] strains varied from the VP8*
sequences of these strains (Fig. 1). These sequence comparisons
suggest that the amino acid sequence of the VP8* glycan binding
domain of P[11] rotavirus strains is distinct from that of the other
rotavirus strains whose glycan partners have been characterized
and that VP8* P[11] could potentially bind previously uncharac-
terized glycan partners.
VP8* P[11] binds glycans with Gal1-4GlcNAc. Potential
glycan partners for VP8* P[11] were screened using a glycan array
comprising 600 glycans. GST-VP8* P[11] showed distinct bind-
ing to a number of glycans, including structures typically found as
both N- and O-linked glycans. Overall, the highest rank was seen
for glycans containing the Gal1-4GlcNAc motif (N-acetyllac-
tosamine; also called LacNAc), in particular, for glycans with mul-
tiple units of LacNAc (polylactosamine). Binding to polylac-
tosamine glycans was seen for GST-VP8* from all P[11] viruses
tested, including N155, N1509, and B223 (Table 1; see Table S1
and Fig. S1 in the supplemental material). Previously known ro-
tavirus binding partners, such as GM1 and GD1a-like glycans,
showed much lower binding (Table 1). The GST-VP8* P[11]
bound with high affinity to the polylactosamine of N-glycans with
no preference for the number of branches (Table 1). The strength
of binding was, however, directly related to the length of the
polylactosamine and less dependent on the terminal monosaccha-
ride. As seen in Table 1, GST-VP8* P[11] bound with less affinity
to the shorter, linear glycans on the array. The addition of other
monosaccharides to polylactosamine had marginal effects on
binding. As can be seen from the glycans listed in Table 1, the
presence of an -1,2-linked fucose (H type II glycan) resulted in
an increase in the strength of binding to glycans with shorter
polylactosamine chains, whereas the presence of Sia (Neu5Ac,
-2,3, or -2,6 linkage) resulted in a reduction in binding
strength. In addition to N-glycans, strong binding to polylac-
tosamine was also seen, which is typical of O-glycans. Thus, the
glycan array results suggest that VP8*P[11] can bind to extended
polylactosamine glycans, which are the precursor for H type II
HBGAs (1). H type II HBGAs are formed by the addition of a
terminal fucose to LacNAc through the activity of -1,2-fucosyl-
transferase. The glycan array results also indicate that VP8*P[11]
can bind H type II derivatives, while binding is clearly reduced by
the addition of Sia to LacNAc. Binding to polylactosamine glycans
was not seen for GST-VP8* from SA11-4F or Wa (see Table S1 and
Fig. S1 in the supplemental material). GST by itself does not bind
to the glycans on the array (data not shown).
GST-VP8* P[11] hemagglutinates adult and cord blood
erythrocytes. H type II glycans are present on the surface of O-
type RBCs and serve as the precursor for the synthesis of A- and
B-type blood group antigens through the activity of glycosyltrans-
ferases (1). Thus, hemagglutination assays serve as a useful tool to
assess the specificity and biological significance of the glycan array
data. In hemagglutination assays with RBCs from adults, GST-
VP8* from the P[11] strains as well as SA11-4F hemagglutinated
type O, type A, and type B RBCs. The Norwalk virus VLP control
TABLE 1 GST-VP8* P[11] shows the highest binding to glycans containing Gal1-4GlcNAc
a
Glycan
Chart
no.
No. of LacNAc
residues
Average
rank
b
Gal1-4GlcNAc1-3Gal1-4GlcNAc1-3Gal1-4GlcNAc1-3Gal1-4GlcNAc1-3Gal1-4GlcNAc1-
2Man1-6(Gal1-4GlcNAc1-3Gal1-4GlcNAc1-3Gal1-4GlcNAc1-3Gal1-4GlcNAc1-
3Gal1-4GlcNAc1-2Man1-3)Man1-4GlcNAc1-4(Fuc1-6)GlcNAc-Sp19
582 6 83.18
Gal1-4GlcNAc1-3Gal1-4GlcNAc1-3Gal1-4GlcNAc1-3Gal1-4GlcNAc1-3Gal1-4GlcNAc1-
3Gal1-4GlcNAc1-2Man1-6(Gal1-4GlcNAc1-3Gal1-4GlcNAc1-3Gal1-4GlcNAc1-
3Gal1-4GlcNAc1-3Gal1-4GlcNAc1-3Gal1-4GlcNAc1-2Man1-3)Man1-4GlcNAc1-
4GlcNAc-Sp25
569 6 78.09
Gal1-4GlcNAc1-3Gal1-4GlcNAc1-3Gal1-4GlcNAc1-3Gal1-4GlcNAc1-3Gal1-4GlcNAc1-
2Man1-6(Gal1-4GlcNAc1-3Gal1-4GlcNAc1-3Gal1-4GlcNAc1-3Gal1-4GlcNAc1-
3Gal1-4GlcNAc1-2Man1-3)Man1-4GlcNAc1-4GlcNAc-Sp25
566 6 77.88
Gal1-4GlcNAc1-3Gal1-4GlcNAc1-3Gal1-4GlcNAc-Sp0 163 3 9.03
Fuc1-2Gal1-4GlcNAc1-3Gal1-4GlcNAc1-3Gal1-4GlcNAc-Sp0 (H type II) 75 3 (terminal fucose) 25.61
Neu5Ac2- 3Gal1-4GlcNAc1-3Gal1-4GlcNAc1-3Gal1-4GlcNAc-Sp0 259 3 (terminal Sia) 5.60
Neu5Ac2-6Gal1-4GlcNAc1-3Gal1-4GlcNAc1-3Gal1-4GlcNAc-Sp0 333 3 (terminal Sia) 3.27
Neu5Ac2-3Gal1-3GalNAc1-4(Neu5Ac2-8Neu5Ac2-3)Gal1-4Glc-Sp0 (GD1a-like) 413 0 0.95
Gal1-3GalNAc1-4(Neu5Ac2-3)Gal1-4Glc-Sp0 (GM1) 145 0 0.59
a
The strength of binding to glycans with Gal1-4GlcNAc (in boldface and underlined) was much higher than that of GM1- and GD1a-like glycans that are known partners for
some rotavirus strains. The raw data from the glycan array experiments are provided in Table S1 and Fig. S1 in the supplemental material.
b
Average rank of representative glycans for binding with GST-VP8* P[11] from human neonatal strain N155 calculated from 4 different arrays. The rank for each glycan was
calculated using the formula 100 (test glycan RFU/highest number of RFU in that assay).
Ramani et al.
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hemagglutinated type O and A RBCs but not type B RBCs, while
GST-VP8* P[14] from HAL1166 hemagglutinated only type A
RBCs, as previously shown (7,31). No hemagglutination was ob-
served with GST-VP8* Wa or the GST control (Table 2). How-
ever, glycan expression on RBCs is developmentally regulated (1).
Unbranched polylactosamine chains are more highly expressed
on the surface of embryonic and cord blood erythrocytes, while
the adult levels of branched chains are reached by about 18
months of age. Therefore, hemagglutination assays were also
carried out with pooled umbilical cord blood erythrocytes.
GST-VP8* P[11] hemagglutinated umbilical cord blood eryth-
rocytes. Partial hemagglutination of cord blood erythrocytes
was seen at the highest concentration of Norwalk virus VLPs,
while GST-VP8* P[14], GST-VP8* Wa, and the GST controls
showed no hemagglutination (Table 2). These data are consistent
with the findings of the glycan array that VP8* P[11] can interact
with H type II glycans on cell surfaces.
The VP8* from the SA11-4F and P[11] strains binds to differ-
ent glycans on RBCs for hemagglutination. SA11-4F is known to
bind terminal Sia on cell surfaces, and neuraminidase treatment of
RBCs resulted in a complete loss of hemagglutination. In contrast,
neuraminidase treatment resulted in an increased hemagglutina-
tion titer for VP8* P[11] from N155 and B223 (Table 3), indicat-
ing that in untreated RBCs, Sia may be masking additional sites for
GST-VP8* P[11] binding.
GST-VP8* P[11] binds all CHO cells, but infectivity is en-
hanced with expression of H type II. The specificity and biologi-
cal relevance of glycan array data were further assessed by binding
and infectivity assays on CHO cells that differ in expression of
HBGA. Binding experiments on parental, H-type, and A-type
CHO cells showed similar binding patterns using GST-VP8*
P[11] (Fig. 2). There was no significant difference in the expres-
sion of polylactosamines between these cell lines (Fig. 3). How-
ever, a significant increase in human and bovine G10P[11] titer
was observed in all CHO cells that express the enzyme Fut2, in-
cluding the double-transfectant CHO cells that express Fut2 and
A- or B-type glycosyltransferases, compared to that observed in
the parental cells (Fig. 4). The enzyme Fut2 catalyzes the addition
of a terminal fucose in -1,2 linkage to the LacNAc precursor. The
additional modification of the H type II to A-type or B-type
HBGA did not result in any further significant increase in virus
titer, indicating that the expression of the H type II was the critical
factor for increased infectivity. Terminal Sia binding animal rota-
virus strain SA11-4F, internal Sia binding Wa, and A-type HBGA
binding HAL1166 were included as controls in the infectivity ex-
periments. No difference in infectivity between the cell lines was
observed for SA11-4F and Wa, while HAL1166 showed signifi-
cantly enhanced infectivity on A-type cells. These data confirm
that the interaction of VP8* P[11] with type II glycans on cell
surfaces is biologically relevant.
Neuraminidase treatment of cells results in enhanced bind-
ing on CHO cells. CHO cells express a large number of sialylated
LacNAc glycans (32). Removal of the terminal Sia should there-
fore result in increased binding on these cells. Indeed, neuramin-
idase treatment of CHO cells resulted in significantly enhanced
binding for GST-VP8* P[11], while no significant difference was
observed with GST-VP8* P[14] (Fig. 5).
Bovine rotavirus-derived VP4 is critical for increased infec-
tivity. The VP4 in the human neonatal P[11] rotaviruses are of
bovine rotavirus origin (20,24). To further confirm that the in-
creased infectivity with H type II CHO cells was directly mediated
by the bovine rotavirus-derived P[11] VP4, infectivity assays were
carried out on parental and single-transfectant H type II CHO
cells using SA11-4F, bovine G10P[11] rotavirus B223, and six re-
assortants of these two parental viruses. B223 shows nearly 94%
identity in the VP4 gene with the VP4 of the bovine-human reas-
sortant G10P[11] strains. Infections with B223 and reassortant
viruses R-491, R-004, and R-198 that express the bovine P[11]
VP4 resulted in significantly higher virus titers in cell lines ex-
pressing H type II than in parental cells (Fig. 6), confirming that
the bovine-origin P[11] VP4 is the key mediator of this virus-cell
interaction.
DISCUSSION
Rotavirus infections in neonates are clinically and epidemiologi-
cally distinct from infections in older children (11). They are
caused by unusual virus strains that appear to be geographically
restricted and show remarkable strain stability in comparison to
the diversity of strains seen in older children. It has been specu-
lated that the age-dependent restriction of unusual viruses for
neonates could be mediated by the interaction of the spike protein
with maturation-dependent host components (33); however,
TABLE 2 GST-VP8* P[11] hemagglutinates adult and cord blood erythrocytes RBCs
Blood type or
source
Reciprocal HA titer
a
SA11-4F Wa HAL1166 N155 N1509 B223 GST NV VLPs
O128 0 0 128 128 128 0 128
A1 128 0 128 128 128 128 0 128
A2 128 0 128 128 128 64 0 128
B6400128 128 64 0 0
Umbilical cord 128 0 0 128 128 128 0 0
a
Reciprocal hemagglutination (HA) titers of adult human type O, type A, and type B RBCs as well as pooled cord blood erythrocytes by GST-VP8* proteins. No hemagglutination
is indicated by 0. Recombinant Norwalk virus (NV) VLPs, HAL1166 GST-VP8* P[14], and GST were included as controls. GST-VP8* P[11] hemagglutinated all RBCs tested.
TABLE 3 Neuraminidase treatment of RBCs results in increased
hemagglutination with GST-VP8* P[11]
Virus strain
Reciprocal HA titer
a
Without neuraminidase With neuraminidase
SA11-4F 512 0
N155 512 2,048
B223 256 2,048
HAL1166 0 0
a
Reciprocal hemagglutination (HA) titers of adult human type O RBCs by GST-VP8*
proteins following neuraminidase treatment. No hemagglutination is indicated by 0.
Neonatal Rotavirus VP8* Binds Type II Precursor
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there has been little conclusive evidence for this to date. This is the
first report demonstrating that the VP8* of a clinically important
neonatal rotavirus strain binds to Gal1-4GlcNAc, the precursor
for type II glycans, and that this binding is biologically relevant for
infection. The binding of P[11] VP8* to precursor glycans raises
important questions about how modification of glycans during
the course of neonatal development may alter susceptibility to
infectious agents.
The repertoire of studies on microbes and their glycan partners
has expanded with recent technological advances in the field of
glycobiology. There is increasing evidence to suggest that glycan-
pathogen interactions play a key role in the pathogenesis of several
infectious agents. This study raises additional questions on
whether developmental modification of glycans may also influ-
ence pathogenesis. Studies in rodents have demonstrated that gly-
cosylation changes in the gut occur throughout the postnatal
phase of development and are controlled by hormonal and dietary
factors through the activity of glycosyltransferases (34). However,
glycan changes in the human neonatal gut have not been exten-
sively studied. Some studies on the developmental regulation of
glycans on human RBCs show that embryonic RBCs have an
abundance of unbranched polylactosamine chains and a relative
lack of branched chains (1). Linear polylactosamine chains are
modified by the activity of the enzyme 1-6 N-acetyl-
glucosaminyltransferase, which transfers N-acetylglucosamine in
1-6 linkage to internal galactose residues in polylactosamine.
This results in the formation of 1-6 N-acetylglucosamine
branches, which may then serve as the substrate for subsequent
LacNAc molecules, thereby forming branched polylactosamine
chains. Adult levels of these branched structures are achieved at
FIG 2 GST-VP8* P[11] binds all CHO cell types. (A and B) Binding of FITC-GST-VP8* P[11] and P[14], respectively, to parental CHO cells (H/A/B, red),
the single-transfectant cells expressing the Fut2 enzyme (H/A/B, purple), and the double-transfectant cells with both Fut2 and A-type glycosyltransferase
(H/A/B, blue).
FIG 3 CHO cells do not differ in the levels of polylactosamine. Parental and H-type CHO cells were assayed for polylactosamine by flow cytometry using LEL
and STL lectins. (A and B) Parental H/A/BCHO cells assayed using LEL and STL lectins, respectively; (C and D) H-type H/A/BCHO cells assayed
using LEL and STL lectins, respectively.
Ramani et al.
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about 18 months of age, while unbranched polylactosamine reac-
tivity reaches very low levels. Little is known about such modifi-
cations in the human gut during the course of development. It is
possible that the polylactosamine is abundantly present in the
neonatal gut but is modified during development by the addition
of various glycans that results in changes in glycan length or degree
of branching. This may in part explain the increased neonatal
susceptibility to P[11] rotaviruses and a reduction in the infectiv-
ity of these strains beyond the neonatal age group. Interestingly,
the globally prevalent human rotavirus strains with P[4] and P[8]
VP4 types that infect older children have been identified to bind to
the modified HBGAs and not the precursor units (8,9).
The hemagglutination assays, binding studies, and differential
infectivity on CHO cells corroborate the findings of the glycan
array. GST-VP8* P[11] hemagglutinates both umbilical cord
blood and adult RBCs due to its ability to bind both polylac-
tosamine glycans and H type II HBGA. Neuraminidase treatment
of RBCs results in increased hemagglutination with GST-VP8*
P[11], whereas a complete loss of hemagglutination was seen with
GST-VP8* from SA11-4F on these cells, indicating that Sia masks
or sterically hinders GST-VP8* P[11] binding to untreated RBCs.
In the CHO cell binding experiments, the neonatal G10P[11] vi-
rus was able to bind all cell types tested, including parental cells;
however, the infectivity was enhanced with the expression of H
type II HBGA. The limited infectivity on the parental CHO cells
may not be due to the lack of availability of the LacNAc glycans in
these cells. It has been demonstrated that the parental CHO cells
express an abundance of LacNAc glycans; however, there are few
glycans with greater than 3 repeats, and CHO cells grown on a
monolayer also express a large number of sialylated LacNAc gly-
cans (32). Removal of sialic acid by neuraminidase treatment re-
sults in increased binding on these cells. In addition, lectin bind-
ing studies on the parental and transfected CHO cells showed that
polylactosamine levels did not change between the parental and
transfected cells. This suggests that the increased infectivity ob-
served in the transfected cells is due to the addition of H type II
structures and not due to concomitant modifications in other gly-
can motifs, such as Sia or LacNAc repeats, that could have oc-
curred due to competition between the -1,2-fucosyltransferase
and other glycosyltransferases. As can be seen from Table 1, the
best binding of GST-VP8* P[11] occurs in the presence of multi-
ple units of LacNAc, and terminal sialylation of LacNAc chains
FIG 4 Infectivity of P[11] rotavirus increases with expression of H antigen. The infectivity of rotaviruses in CHO cell lines genetically modified to stably express
different glycans is shown. Infection was carried out in parental CHO cells (H/A/B), the single-transfectant cells expressing the Fut2 enzyme (H/A/
B), and the double-transfectant cells with both Fut2 and A type glycosyltransferase (H/A/B) or B type glycosyltransferase (H/A/B). The rotavirus
strains used (and their titers on CHO cells) include SA11-4F, which binds terminal Sia (4 105 FFU/ml, where FFU is focus-forming units); Wa, which binds
internal Sia (5.5 105 FFU/ml); HAL1166, which binds A-type HBGA (7.7 103 FFU/ml); human neonatal P[11] rotavirus strain N1509 (1.5 104 FFU/ml);
and bovine P[11] rotavirus strain B223 (5.2 103 FFU/ml). The yaxis represents the fold difference in comparison to the results for the parental cells. Error bars
represent standard errors of the means of data from quadruplicate wells from at least 3 independent experiments. A Pvalue of 0.001 was considered statistically
significant and is represented by an asterisk.
FIG 5 Neuraminidase treatment of CHO cells results in increased binding to
GST-VP8* P[11]. Binding of FITC-GST-VP8* P[11] or P[14] on H/A/B,
H/A/B, and H/A/BCHO cells was carried out in duplicate with
and without neuraminidase treatment. The change in the median fluorescence
intensity of binding was compared between untreated and neuraminidase-
treated cell lines and expressed as the percent change compared to the results
for untreated cells. The data from all cell lines were pooled for each protein and
treatment group. Error bars represent the standard error of mean change in
binding for all experiments within a group. A Pvalue of 0.05 was considered
statistically significant and is represented by an asterisk.
Neonatal Rotavirus VP8* Binds Type II Precursor
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results in a reduction in the strength of binding to GST-VP8*
P[11] compared to that for fucosylated LacNAc (H type II). It
should be noted that long polylactosamines terminating with fu-
cose or Sia are not present in the glycan array, and this precludes
making a definitive assessment of the strength of their binding to
GST-VP8* P[11] in the array compared to that of large polylac-
tosamine glycans lacking these monosaccharides.
In this study, it was seen that bovine rotavirus G10P[11] B223
showed a glycan binding profile and hemagglutination properties
similar to those of the human neonatal viruses. G10P[11] rotavi-
rus infections have been widely reported to cause diarrhea in cattle
in many parts of the world (13,15,35,36). These strains contain
all gene segments of bovine rotavirus origin. The human neonatal
G10P[11] infections are, however, caused by bovine-human reas-
sortant strains; these viruses possess a VP4 of bovine rotavirus
origin (20,24). To confirm that the presence of the bovine P[11]
VP4 was key for the increased infectivity on H type II CHO cells,
infectivity assays were carried out with a bovine G10P[11] rotavi-
rus strain B223 and reassortants derived using B223 and SA11-4F
that differ in their genetic makeup. B223 and all reassortant vi-
ruses possessing the bovine P[11] VP4 (R-491, R-004, and R-198)
showed increased infectivity on H type II cells. The availability of
reasssortant rotavirus strains thus allowed confirmation that the
P[11] VP4 of bovine rotavirus origin is the key mediator of the
interaction with type II glycans described in this study. It should,
however, be noted that the magnitude of the fold change was the
highest for B223 in comparison to that for the reassortant viruses,
indicating a role for other genes and gene combinations in infec-
tivity.
The findings of this study are highly relevant in the context
of current rotavirus vaccines in developing countries. Rotavi-
rus diarrhea results in nearly half a million deaths annually;
over 80% of these deaths occur in the developing countries of
Asia and Africa (37). Currently licensed vaccines that are highly
efficacious in developed countries do not appear to be as effec-
tive in the developing world (3840). This has led to the eval-
uation of new vaccines and alternate immunization schedules,
including maternal and neonatal immunizations. One of the
new vaccines being evaluated in India is 116E, which is based
on an asymptomatic human neonatal G9P[11] rotavirus. This
strain also possesses a P[11] VP4 gene of bovine rotavirus ori-
gin and has been found to be highly immunogenic (21,24). The
findings of this study suggest that neonatal immunization with
this neonatal P[11] virus may prove to be effective, as these
strains may be able to replicate more efficiently in the neonatal
gut and thus result in better vaccine take. However, this also
leads to questions of whether vaccine uptake and response may
differ at different ages of vaccination depending on the VP4
genotype of the vaccine virus and glycan profile of the vacci-
nated infant. These questions will be addressed through future
studies involving characterization of glycan interactions for
additional rotavirus VP4 types, including the globally preva-
lent genotypes and those present in vaccine viruses.
ACKNOWLEDGMENTS
We thank Monica McNeal, Cincinnati Children’s Hospital and Medical
Center, Cincinnati, OH, for help with adapting the human neonatal
G10P[11] virus to cell culture. We also thank Jézabel Rocher, INSERM,
Université de Nantes, Nantes, France, for her expertise with the prepara-
tion of CHO transfectants and flow cytometry analysis.
This study was supported by NIH grants R01 AI080656, R01 AI36040,
and P30 DK56338, which funds the Texas Medical Center Digestive Dis-
FIG 6 Increased infectivity on H-antigen-expressing CHO cells is mediated by VP4. The infectivity of SA11, B223, and laboratory-derived reassortant rotaviruses
in parental CHO cells (H/A/B) and cells expressing H antigen (H/A/B) is shown. The origins of the VP4, VP7, and remaining genes in virus strains
are listed below the strain. Pvalues of 0.001 represent significant differences in virus infectivity between the parental and single-transfectant cell lines and are
represented by asterisks.
Ramani et al.
7262 jvi.asm.org Journal of Virology
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eases Center, and a grant from Robert Welch Foundation (Q1279). Gly-
can array analyses were provided by the Protein-Glycan Interaction Re-
source of the Center for Functional Glycomics, which was supported by
GM62116 and GM98791. Flow cytometry studies were carried out at the
Cytometry and Cell Sorting Core at the Baylor College of Medicine with
funding from the NIH (NIAID P30AI036211, NCI P30CA125123, and
NCRR S10RR024574) and the assistance of Joel M. Sederstrom.
REFERENCES
1. Stanley P, Cummings RD. 2009. Structures common to different glycans,
p 175–198. In Varki A, Cummings RD, Esko JD, Freeze HH, Stanley P,
Bertozzi CR, Hart GW, Etzler ME (ed), Essentials in glycobiology, 2nd ed.
Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.
2. Blanchard H, Yu X, Coulson BS, von Itzstein M. 2007. Insight into host
cell carbohydrate-recognition by human and porcine rotavirus from crys-
tal structures of the virion spike associated carbohydrate-binding domain
(VP8*). J. Mol. Biol. 367:1215–1226.
3. Ciarlet M, Estes MK. 1999. Human and most animal rotavirus strains do
not require the presence of sialic acid on the cell surface for efficient in-
fectivity. J. Gen. Virol. 80(Pt 4):943–948.
4. Dormitzer PR, Sun ZY, Wagner G, Harrison SC. 2002. The rhesus
rotavirus VP4 sialic acid binding domain has a galectin fold with a novel
carbohydrate binding site. EMBO J. 21:885– 897.
5. Monnier N, Higo-Moriguchi K, Sun ZY, Prasad BV, Taniguchi K,
Dormitzer PR. 2006. High-resolution molecular and antigen structure of
the VP8* core of a sialic acid-independent human rotavirus strain. J. Virol.
80:1513–1523.
6. Haselhorst T, Fleming FE, Dyason JC, Hartnell RD, Yu X, Holloway G,
Santegoets K, Kiefel MJ, Blanchard H, Coulson BS, von Itzstein M.
2009. Sialic acid dependence in rotavirus host cell invasion. Nat. Chem.
Biol. 5:91–93.
7. Hu L, Crawford SE, Czako R, Cortes-Penfield NW, Smith DF, Le Pendu
J, Estes MK, Prasad BVV. 2012. Cell attachment protein VP8* of a
human rotavirus specifically interacts with A-type histo-blood group an-
tigen. Nature 485:256 –259.
8. Huang P, Xia M, Tan M, Zhong W, Wei C, Wang L, Morrow A, Jiang
X. 2012. Spike protein VP8* of human rotavirus recognizes histo-blood
group antigens in a type-specific manner. J. Virol. 86:4833– 4843.
9. Liu Y, Huang P, Tan M, Biesiada J, Meller J, Castello AA, Jiang B, Jiang
X. 2012. Rotavirus VP8*: phylogeny, host range and interaction with
HBGAs. J. Virol. 86:9899 –9910.
10. Flores J, Sears J, Green KY, Perez-Schael I, Morantes A, Daoud G,
Gorziglia M, Hoshino Y, Chanock RM, Kapikian AZ. 1988. Genetic
stability of rotaviruses recovered from asymptomatic neonatal infections.
J. Virol. 62:4778 – 4781.
11. Haffejee IE. 1991. Neonatal rotavirus infections. Rev. Infect. Dis. 13:957–
962.
12. Estes MK, Kapikian AZ. 2007. Rotaviruses, p 1917–1974. In Knipe
DM, Howley PM, Griffin DE, Lamb RA, Martin MA, Roizman B, Straus
SE (ed), Fields virology, 5th ed. Lippincott Williams & Wilkins, Phil-
adelphia, PA.
13. Varshney B, Jagannath MR, Vethanayagam RR, Kodhandharaman S,
Jagannath HV, Gowda K, Singh DK, Rao CD. 2002. Prevalence of, and
antigenic variation in, serotype G10 rotaviruses and detection of serotype
G3 strains in diarrheic calves: implications for the origin of G10P11 or P11
type reassortant asymptomatic strains in newborn children in India. Arch.
Virol. 147:143–165.
14. Vethanayagam RR, Ananda Babu M, Nagalaxmi KS, Maiya PP, Ven-
katesh HA, Purohit S, Behl R, Bhan MK, Ward RL, Greenberg HB,
Durga Rao C. 2004. Possible role of neonatal infection with the asymp-
tomatic reassortant rotavirus (RV) strain I321 in the decrease in hospital
admissions for RV diarrhea, Bangalore, India, 1988-1999. J. Infect. Dis.
189:2282–2289.
15. Gulati BR, Nakagomi O, Koshimura Y, Nakagomi T, Pandey R. 1999.
Relative frequencies of G and P types among rotaviruses from Indian
diarrheic cow and buffalo calves. J. Clin. Microbiol. 37:2074 –2076.
16. Ramani S, Sowmyanarayanan TV, Gladstone BP, Bhowmick K, Asir-
vatham JR, Jana AK, Kuruvilla KA, Kumar M, Gibikote S, Kang G.
2008. Rotavirus infection in the neonatal nurseries of a tertiary care hos-
pital in India. Pediatr. Infect. Dis. J. 27:719 –723.
17. Sowmyanarayanan TV, Ramani S, Sarkar R, Arumugam R, Warier JP,
Moses PD, Simon A, Agarwal I, Bose A, Arora R, Kang G. 2012. Severity
of rotavirus gastroenteritis in Indian children requiring hospitalization.
Vaccine 30(Suppl 1):A167–A172.
18. Banerjee I, Gladstone BP, Le Fevre AM, Ramani S, Iturriza-Gomara M,
Gray JJ, Brown DW, Estes MK, Muliyil JP, Jaffar S, Kang G. 2007.
Neonatal infection with G10P[11] rotavirus did not confer protection
against subsequent rotavirus infection in a community cohort in Vellore,
South India. J. Infect. Dis. 195:625– 632.
19. Gladstone BP, Ramani S, Mukhopadhya I, Muliyil J, Sarkar R, Rehman
AM, Jaffar S, Gomara MI, Gray JJ, Brown DW, Desselberger U, Craw-
ford SE, John J, Babji S, Estes MK, Kang G. 2011. Protective effect of
natural rotavirus infection in an Indian birth cohort. N. Engl. J. Med.
365:337–346.
20. Ramani S, Iturriza-Gomara M, Jana AK, Kuruvilla KA, Gray JJ, Brown
DW, Kang G. 2009. Whole genome characterization of reassortant
G10P[11] strain (N155) from a neonate with symptomatic rotavirus in-
fection: identification of genes of human and animal rotavirus origin. J.
Clin. Virol. 45:237–244.
21. Bhandari N, Sharma P, Taneja S, Kumar T, Rongsen-Chandola T,
Appaiahgari MB, Mishra A, Singh S, Vrati S. 2009. A dose-escalation
safety and immunogenicity study of live attenuated oral rotavirus vaccine
116E in infants: a randomized, double-blind, placebo-controlled trial. J.
Infect. Dis. 200:421– 429.
22. Cicirello HG, Das BK, Gupta A, Bhan MK, Gentsch JR, Kumar R, Glass
RI. 1994. High prevalence of rotavirus infection among neonates born at
hospitals in Delhi, India: predisposition of newborns for infection with
unusual rotavirus. Pediatr. Infect. Dis. J. 13:720 –724.
23. Das BK, Gentsch JR, Hoshino Y, Ishida S, Nakagomi O, Bhan MK,
Kumar R, Glass RI. 1993. Characterization of the G serotype and geno-
group of New Delhi newborn rotavirus strain 116E. Virology 197:99 –107.
24. Gentsch JR, Das BK, Jiang B, Bhan MK, Glass RI. 1993. Similarity of the
VP4 protein of human rotavirus strain 116E to that of the bovine B223
strain. Virology 194:424 – 430.
25. Kang G, Arora R, Chitambar SD, Deshpande J, Gupte MD, Kulkarni M,
Naik TN, Mukherji D, Venkatasubramaniam S, Gentsch JR, Glass RI,
Parashar UD. 2009. Multicenter, hospital-based surveillance of rotavirus
disease and strains among Indian children aged 5 years. J. Infect. Dis.
200(Suppl 1):S147–S153.
26. Alvarez RA, Blixt O. 2006. Identification of ligand specificities for glycan
binding proteins using glycan arrays, p. In Fukuda M (ed), Glycobiology.
Elsevier Academic Press, San Diego, CA.
27. Thorpe SJ, Boult CE, Stevenson FK, Scott ML, Sutherland J, Spellerberg
MB, Natvig JB, Thompson KM. 1997. Cold agglutinin activity is com-
mon among human monoclonal IgM Rh system antibodies using the
V4-34 heavy chain variable gene segment. Transfusion 37:1111–1116.
28. Guillon P, Clement M, Sebille V, Rivain JG, Chou CF, Ruvoen-Clouet
N, Le Pendu J. 2008. Inhibition of the interaction between the SARS-CoV
spike protein and its cellular receptor by anti-histo-blood group antibod-
ies. Glycobiology 18:1085–1093.
29. Chen D, Burns JW, Estes MK, Ramig RF. 1989. Phenotypes of rotavirus
reassortants depend upon the recipient genetic background. Proc. Natl.
Acad. Sci. U. S. A. 86:3743–3747.
30. Chen DY, Estes MK, Ramig RF. 1992. Specific interactions between
rotavirus outer capsid proteins VP4 and VP7 determine expression of a
cross-reactive, neutralizing VP4-specific epitope. J. Virol. 66:432– 439.
31. Hutson AM, Atmar RL, Marcus DM, Estes MK. 2003. Norwalk virus-
like particle hemagglutination by binding to H histo-blood group anti-
gens. J. Virol. 77:405– 415.
32. North SJ, Huang HH, Sundaram S, Jang-Lee J, Etienne AT, Trollope A,
Chalabi S, Dell A, Stanley P, Haslam SM. 2010. Glycomics profiling of
Chinese hamster ovary cell glycosylation mutants reveals N-glycans of a
novel size and complexity. J. Biol. Chem. 285:5759 –5775.
33. Palombo EA, Bishop RF. 1994. Genetic analysis of NSP1 genes of human
rotaviruses isolated from neonates with asymptomatic infection. J. Gen.
Virol. 75(Pt 12):3635–3639.
34. Biol-N=garagba MC, Louisot P. 2003. Regulation of the intestinal glyco-
protein glycosylation during postnatal development: role of hormonal
and nutritional factors. Biochimie 85:331–352.
35. Fukai K, Maeda Y, Fujimoto K, Itou T, Sakai T. 2002. Changes in the
prevalence of rotavirus G and P types in diarrheic calves from the Ka-
goshima Prefecture in Japan. Vet. Microbiol. 86:343–349.
36. Reidy N, Lennon G, Fanning S, Power E, O’Shea H. 2006. Molecular
characterisation and analysis of bovine rotavirus strains circulating in Ire-
land 2002-2004. Vet. Microbiol. 117:242–247.
Neonatal Rotavirus VP8* Binds Type II Precursor
July 2013 Volume 87 Number 13 jvi.asm.org 7263
on June 13, 2014 by guesthttp://jvi.asm.org/Downloaded from
37. Parashar UD, Burton A, Lanata C, Boschi-Pinto C, Shibuya K, Steele D,
Birmingham M, Glass RI. 2009. Global mortality associated with rotavirus
disease among children in 2004. J. Infect. Dis. 200(Suppl 1):S9 –S15.
38. Armah GE, Sow SO, Breiman RF, Dallas MJ, Tapia MD, Feikin DR,
Binka FN, Steele AD, Laserson KF, Ansah NA, Levine MM, Lewis K,
Coia ML, Attah-Poku M, Ojwando J, Rivers SB, Victor JC, Nyambane
G, Hodgson A, Schodel F, Ciarlet M, Neuzil KM. 2010. Efficacy of
pentavalent rotavirus vaccine against severe rotavirus gastroenteritis in
infants in developing countries in sub-Saharan Africa: a randomised, dou-
ble-blind, placebo-controlled trial. Lancet 376:606 – 614.
39. Madhi SA, Cunliffe NA, Steele D, Witte D, Kirsten M, Louw C, Ngwira
B, Victor JC, Gillard PH, Cheuvart BB, Han HH, Neuzil KM. 2010.
Effect of human rotavirus vaccine on severe diarrhea in African infants. N.
Engl. J. Med. 362:289 –298.
40. Zaman K, Dang DA, Victor JC, Shin S, Yunus M, Dallas MJ, Podder G,
Vu DT, Le TP, Luby SP, Le HT, Coia ML, Lewis K, Rivers SB, Sack DA,
Schodel F, Steele AD, Neuzil KM, Ciarlet M. 2010. Efficacy of pentava-
lent rotavirus vaccine against severe rotavirus gastroenteritis in infants in
developing countries in Asia: a randomised, double-blind, placebo-
controlled trial. Lancet 376:615– 623.
Ramani et al.
7264 jvi.asm.org Journal of Virology
on June 13, 2014 by guesthttp://jvi.asm.org/Downloaded from

Supplementary resource (1)

... Preincubation with exogenous Neu5Ac, Neu5Gc, and α-Gal prevented infections by P [5]-bearing strains in MA104 cells, suggesting that both sialic acids and α-Gal epitopes are recognized by VP8* of P [5] RVs [35]. The VP8*s of G10P [11] HRV N155 and BRV B223 have been reported to bind to non-sialylated glycans containing Galβ-GlcNAc, and the glycan array studies have shown that sialic acid is not required for binding of these VP8*s [32,36,37]. Here, we report that VP8* of P [11] PRV 4555 recognizes not only type II precursors of H-type antigen (Galβ1,4-GlcNAcβ1,3-Galβ1,4-Glc) but also interacts explicitly with gangliosides containing subterminal sialic acids. ...
... Although it is known that VP8* proteins of P [11] RVs recognize precursors of H-type antigens [31,36,37], it is unknown whether P [11] RVs may additionally use other glycans or proteins as attachment factors or receptors. Sialic acids have been shown to serve as receptor determinants for several (mainly animal) RVs [28], rendering them NA-sensitive. ...
... Nevertheless, it is a well-accepted paradigm that NA-resistant rotaviruses do not use sialic acid for host cell recognition [3]. All viruses of the P [11] genotype are NA-resistant and, therefore, are expected not to interact with sialic acid [32,37]. ...
Article
Full-text available
Rotaviruses, non-enveloped viruses with a double-stranded RNA genome, are the leading etiological pathogen of acute gastroenteritis in young children and animals. The P[11] genotype of rotaviruses exhibits a tropism for neonates. In the present study, a binding assay using synthetic oligosaccharides demonstrated that the VP8* protein of P[11] porcine rotavirus (PRV) strain 4555 binds to lacto-N-neotetraose (LNnT) with the sequence Galβ1,4-GlcNAcβ1,3-Galβ1,4-Glc, one of the core parts of histo-blood group antigen (HBGA) and milk glycans. However, infections were significantly inhibited by blocking of endogenous monosialoganglioside (GM) GM1a with cholera toxin B subunit and preincubation of the virus with exogenous GM1a, suggesting that GM1a is involved in the infection of P[11] PRV 4555. In addition to GM1a, preincubation of the virus with exogenous disialogangliosides (GD) GD1a, GD1b, and trisialoganglioside (GT) GT1b also prevented infection. In contrast, exogenous ganglioside GM3 only inhibited infections at an early time point, and exogenous asyalosphingolipids GA1 and LacCer did not show any inhibitory effect on infections. This indicates that P[11] PRV 4555 preferentially utilizes gangliosides containing subterminal sialic acids. Further experiments revealed that P[11] PRV 4555 infections were prevented by preincubation of the virus with Neu5Ac and Neu5Gc. These results confirmed that sialic acids are essential for P[11] PRV 4555 cell entry, despite the classification as NA-resistant strain. Overall, our results proved that P[11] rotavirus not only binds to the Gal-GlcNAc motif but also utilizes gangliosides containing subterminal sialic acids.
... Current evidence suggests that the two protein components of the outer layer, VP4 and VP7, have distinct functions in DLP delivery [14][15][16][17][18]. VP4, activated by proteolytic cleavage into VP8* and VP5*, is the principal membrane-interacting partner; VP7, held together by Ca 2+ ions at its trimer interfaces, is a Ca 2+ -sensitive "clamp" that anchors VP4 onto the underlying DLP. For mammalian rotaviruses, cell attachment generally requires interaction of the VP8* moiety of VP4 with a glycolipid headgroup [19][20][21][22][23]. For rhesus rotavirus (RRV) entering BSC-1 or SVG-A cells, uptake of the virus particle into a small vesicle does not require clathrin, dynamin, or related activities; the virus particle appears to produce its own engulfment by wrapping the plasma membrane around itself [24]. ...
... We tested whether an interaction of virions with a membrane could lead to Ca 2+ permeability, without contribution of potential cellular factors, using liposomes containing, in their lumen, a soluble, Ca 2+ -sensitive fluorophore (Fluo-4) and, in their bilayer, a fluorescently labelled lipid (Cy5-DOPE) for visualization, as well as biotinylated lipids for attachment to an avidin-coated coverslip. We prepared the liposomes in the absence of Ca 2+ and used a lipid composition similar to that of mammalian-cell plasma membranes supplemented with 2.5% GD1a, a validated rotavirus attachment factor [19][20][21][22][23]. In the absence of Ca 2+ , we established the background fluorescence of the incorporated Fluo-4 dye. ...
Article
Full-text available
Rotaviruses infect cells by delivering into the cytosol a transcriptionally active inner capsid particle (a "double-layer particle": DLP). Delivery is the function of a third, outer layer, which drives uptake from the cell surface into small vesicles from which the DLPs escape. In published work, we followed stages of rhesus rotavirus (RRV) entry by live-cell imaging and correlated them with structures from cryogenic electron microscopy and tomography (cryo-EM and cryo-ET). The virus appears to wrap itself in membrane, leading to complete engulfment and loss of Ca²⁺ from the vesicle produced by the wrapping. One of the outer-layer proteins, VP7, is a Ca²⁺-stabilized trimer; loss of Ca²⁺ releases both VP7 and the other outer-layer protein, VP4, from the particle. VP4, activated by cleavage into VP8* and VP5*, is a trimer that undergoes a large-scale conformational rearrangement, reminiscent of the transition that viral fusion proteins undergo to penetrate a membrane. The rearrangement of VP5* thrusts a 250-residue, C-terminal segment of each of the three subunits outward, while allowing the protein to remain attached to the virus particle and to the cell being infected. We proposed that this segment inserts into the membrane of the target cell, enabling Ca²⁺ to cross. In the work reported here, we show the validity of key aspects of this proposed sequence. By cryo-EM studies of liposome-attached virions ("triple-layer particles": TLPs) and single-particle fluorescence imaging of liposome-attached TLPs, we confirm insertion of the VP4 C-terminal segment into the membrane and ensuing generation of a Ca²⁺ "leak". The results allow us to formulate a molecular description of early events in entry. We also discuss our observations in the context of other work on double-strand RNA virus entry.
... The main targets for RV infection are mature enterocytes -absorptive intestinal epithelial cells (IECs) located at the tips of intestinal villi [14]. Several molecules on enterocytes and other IECs such as integrins [15], sialic acids (SAs) [16], gangliosides [17], N-and O-glycans [18][19][20], heat-shock cognate protein (hsc70) [21] and tight junction proteins [22] are recognized by RV spike protein (VP4) and serve as ligands for RV attachment and entry to IECs [23]. More specifically, histo-blood group antigens (HBGAs) and SA-containing molecules have been demonstrated to serve as attachment factors during RVA and RVC infection [24]. ...
... Black circles represent the significance (p-value < 0.05, i.e. 1.3 in − log10 format)Raev et al. Virology Journal (2023) 20:Change of expression of genes encoding glycosyltransferases catalyzing transfer of sugar residues to glycan cores (glycan core extension) in PIEs infected with RVC, G9P[13] and G5P[7] vs control (non-infected PIEs). The peripheral terminal region of glycan cores may include d-galactose (Gal), N-acetylglucosamine (GlcNAc) and SA residues whose transfer is provided by several glycosyltransferases in a core-specific manner. ...
Article
Full-text available
Background Rotavirus C (RVC) is the major causative agent of acute gastroenteritis in suckling piglets, while most RVAs mostly affect weaned animals. Besides, while most RVA strains can be propagated in MA-104 and other continuous cell lines, attempts to isolate and culture RVC strains remain largely unsuccessful. The host factors associated with these unique RVC characteristics remain unknown. Methods In this study, we have comparatively evaluated transcriptome responses of porcine ileal enteroids infected with RVC G1P[1] and two RVA strains (G9P[13] and G5P[7]) with a focus on innate immunity and virus-host receptor interactions. Results The analysis of differentially expressed genes regulating antiviral immune response indicated that in contrast to RVA, RVC infection resulted in robust upregulation of expression of the genes encoding pattern recognition receptors including RIG1-like receptors and melanoma differentiation-associated gene-5. RVC infection was associated with a prominent upregulation of the most of glycosyltransferase-encoding genes except for the sialyltransferase-encoding genes which were downregulated similar to the effects observed for G9P[13]. Conclusions Our results provide novel data highlighting the unique aspects of the RVC-associated host cellular signalling and suggest that increased upregulation of the key antiviral factors maybe one of the mechanisms responsible for RVC age-specific characteristics and its inability to replicate in most cell cultures.
... The P genotype approach involves the P[11] rotavirus (ROTAVAC strain) which commonly infects neonates. Neonates recognize a type 2 HBGA precursor (Galß1-4GlcNAc) (Liu et al., 2013;Ramani et al., 2013), as a host ligand or receptor. The age at which Galß1-4GlcNAc turns on could be crucial in determining the susceptibility of P[11] in neonates/infants compared to older children. ...
Article
Full-text available
In the present study, first, rotaviruses that caused acute gastroenteritis in children under five years of age during the time before the vaccine was introduced in Iran (1986 to 2023) are reviewed. Subsequently, the antigenic epitopes of the VP7 and VP4/VP8 proteins in circulating rotavirus strains in Iran and that of the vaccine strains were compared and their genetic differences in histo-blood group antigens (HBGAs) and the potential impact on rotavirus infection susceptibility and vaccine efficacy were discussed. Overall data indicate that rotavirus was estimated in about 38.1 % of samples tested. The most common genotypes or combinations were G1 and P[8], or G1P[8]. From 2015 to 2023, there was a decline in the prevalence of G1P[8], with intermittent peaks of genotypes G3P[8] and G9P[8]. The analyses suggested that the monovalent Rotarix vaccine or monovalent vaccines containing the G1P[8] component might be proper in areas with a similar rotavirus genotype pattern and genetic background as the Iranian population where the G1P[8] strain is the most predominant and has the ability to bind to HBGA secretors. While the same concept can be applied to RotaTeq and RotasIIL vaccines, their complex vaccine technology, which involves reassortment, makes them less of a priority. The ROTASIIL vaccine, despite not having the VP4 arm (P[5]) as a suitable protection option, has previously shown the ability to neutralize not only G9-lineage I strains but also other G9-lineages at high titers. Thus, vaccination with the ROTASIIL vaccine may be more effective in Iran compared to RotaTeq. However, considering the rotavirus genotypic pattern, ROTAVAC might not be a good choice for Iran. Overall, the findings of this study provide valuable insights into the prevalence of rotavirus strains and the potential effectiveness of different vaccines in the Iranian and similar populations.
... . Rotarix® (RV1) is administered in 2 doses at ages 2 months and 4 months. Clinical trials of the vaccine detected no increased risk of intussusception.[47][48][49] Rotasiil® (RV5) is administered as 3 doses at 6 weeks, 10 weeks and 14 weeks of age.[55] ...
Article
Full-text available
Hospital Pharmacy is a pharmacy that provides services as part of the hospital organisation and is run by a chemist with a devotion to their vocation and the necessary legal qualifications. I'm working on my assignment in a hospital. We observed a variety of illnesses in patients during our practise session, including rotavirus and measles. Ondansetron and ORS are two medications that are recommended for rotavirus. prescription drugs for measles include ibuprofen, paracetamol, vitamin A supplements, and calamine lotion.
Preprint
Full-text available
Rotaviruses infect cells by delivering into the cytosol a transcriptionally active inner capsid particle (a "double-layer particle": DLP). Delivery is the function of a third, outer layer, which drives uptake from the cell surface into small vesicles from which the DLPs escape. In published work, we followed stages of rhesus rotavirus (RRV) entry by live-cell imaging and correlated them with structures from cryogenic electron microscopy and tomography (cryo-EM and cryo-ET). The virus appears to wrap itself in membrane, leading to complete engulfment and loss of Ca ²⁺ from the vesicle produced by the wrapping. One of the outer-layer proteins, VP7, is a Ca ²⁺ -stabilized trimer; loss of Ca ²⁺ releases both outer-layer proteins from the particle. The other outer-layer protein, VP4, activated by cleavage into VP8* and VP5*, is a trimer that undergoes a large-scale conformational rearrangement, reminiscent of the transition that viral fusion proteins undergo to penetrate a membrane. The rearrangement of VP5* thrusts a 250-residue, C-terminal segment of each of the three subunits outward, while allowing the protein to remain attached to the virus particle and to the cell being infected. We proposed that this segment inserts into the membrane of the target cell, enabling Ca ²⁺ to cross. In the work reported here, we show the validity of key aspects of this proposed sequence. By cryo-EM studies of liposome-attached virions ("triple-layer particles": TLPs) and single-particle fluorescence imaging of liposome-attached TLPs, we confirm insertion of the VP4 C-terminal segment into the membrane and ensuing generation of a Ca ²⁺ "leak". The results allow us to formulate a molecular description of early events in entry. We also discuss our observations in the context of other work on double-strand RNA virus entry.
Article
Full-text available
The distal portion of rotavirus (RV) VP4 spike protein (VP8*) is implicated in binding to cellular receptors, thereby facilitating viral attachment and entry. While VP8* of some animal RVs engage sialic acid, human RVs often attach to and enter cells in a sialic acid-independent manner. A recent study demonstrated that the major human RVs (P[4], P[6], and P[8]) recognize human histo-blood group antigens (HBGAs). In this study, we performed a phylogenetic analysis of RVs and showed further variations of RV interaction with HBGAs. On the basis of the VP8* sequences, RVs are grouped into five P genogroups (P[I] to P[V]), of which P[I], P[IV], and P[V] mainly infect animals, P[II] infects humans, and P[III] infects both animals and humans. The sialic acid-dependent RVs (P[1], P[2], P[3], and P[7]) form a subcluster within P[I], while all three major P genotypes of human RVs (P[4], P[6], and P[8]) are clustered in P[II]. We then characterized three human RVs (P[9], P[14], and P[25]) in P[III] and observed a new pattern of binding to the type A antigen which is distinct from that of the P[II] RVs. The binding was demonstrated by hemagglutination and saliva binding assay using recombinant VP8* and native RVs. Homology modeling and mutagenesis study showed that the locations of the carbohydrate binding interfaces are shared with the sialic acid-dependent RVs, although different amino acids are involved. The P[III] VP8* proteins also bind the A antigens of the porcine and bovine mucins, suggesting the A antigen as a possible factor for cross-species transmission of RVs. Our study suggests that HBGAs play an important role in RV infection and evolution.
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Full-text available
As with many other viruses, the initial cell attachment of rotaviruses, which are the major causative agent of infantile gastroenteritis, is mediated by interactions with specific cellular glycans. The distally located VP8* domain of the rotavirus spike protein VP4 (ref. 5) mediates such interactions. The existing paradigm is that 'sialidase-sensitive' animal rotavirus strains bind to glycans with terminal sialic acid (Sia), whereas 'sialidase-insensitive' human rotavirus strains bind to glycans with internal Sia such as GM1 (ref. 3). Although the involvement of Sia in the animal strains is firmly supported by crystallographic studies, it is not yet known how VP8* of human rotaviruses interacts with Sia and whether their cell attachment necessarily involves sialoglycans. Here we show that VP8* of a human rotavirus strain specifically recognizes A-type histo-blood group antigen (HBGA) using a glycan array screen comprised of 511 glycans, and that virus infectivity in HT-29 cells is abrogated by anti-A-type antibodies as well as significantly enhanced in Chinese hamster ovary cells genetically modified to express the A-type HBGA, providing a novel paradigm for initial cell attachment of human rotavirus. HBGAs are genetically determined glycoconjugates present in mucosal secretions, epithelia and on red blood cells, and are recognized as susceptibility and cell attachment factors for gastric pathogens like Helicobacter pylori and noroviruses. Our crystallographic studies show that the A-type HBGA binds to the human rotavirus VP8* at the same location as the Sia in the VP8* of animal rotavirus, and suggest how subtle changes within the same structural framework allow for such receptor switching. These results raise the possibility that host susceptibility to specific human rotavirus strains and pathogenesis are influenced by genetically controlled expression of different HBGAs among the world's population.
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Rotaviruses (RVs), an important cause of severe diarrhea in children, have been found to recognize sialic acid as receptors for host cell attachment. While a few animal RVs (of P[1], P[2], P[3], and P[7]) are sialidase sensitive, human RVs and the majority of animal RVs are sialidase insensitive. In this study, we demonstrated that the surface spike protein VP8* of the major P genotypes of human RVs interacts with the secretor histo-blood group antigens (HBGAs). Strains of the P[4] and P[8] genotypes shared reactivity with the common antigens of Lewis b (Leb) and H type 1, while strains of the P[6] genotype bound the H type 1 antigen only. The bindings between recombinant VP8* and human saliva, milk, or synthetic HBGA oligosaccharides were demonstrated, which was confirmed by blockade of the bindings by monoclonal antibodies (MAbs) specific to Leb and/or H type 1. In addition, specific binding activities were observed when triple-layered particles of a P[8] (Wa) RV were tested. Our results suggest that the spike protein VP8* of RVs is involved in the recognition of human HBGAs that may function as ligands or receptors for RV attachment to host cells.
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More than 500,000 deaths are attributed to rotavirus gastroenteritis annually worldwide, with the highest mortality in India. Two successive, naturally occurring rotavirus infections have been shown to confer complete protection against moderate or severe gastroenteritis during subsequent infections in a birth cohort in Mexico. We studied the protective effect of rotavirus infection on subsequent infection and disease in a birth cohort in India (where the efficacy of oral vaccines in general has been lower than expected). We recruited children at birth in urban slums in Vellore; they were followed for 3 years after birth, with home visits twice weekly. Stool samples were collected every 2 weeks, as well as on alternate days during diarrheal episodes, and were tested by means of enzyme-linked immunosorbent assay and polymerase-chain-reaction assay. Serum samples were obtained every 6 months and evaluated for seroconversion, defined as an increase in the IgG antibody level by a factor of 4 or in the IgA antibody level by a factor of 3. Of 452 recruited children, 373 completed 3 years of follow-up. Rotavirus infection generally occurred early in life, with 56% of children infected by 6 months of age. Levels of reinfection were high, with only approximately 30% of all infections identified being primary. Protection against moderate or severe disease increased with the order of infection but was only 79% after three infections. With G1P[8], the most common viral strain, there was no evidence of homotypic protection. Early infection and frequent reinfection in a locale with high viral diversity resulted in lower protection than has been reported elsewhere, providing a possible explanation why rotavirus vaccines have had lower-than-expected efficacy in Asia and Africa. (Funded by the Wellcome Trust.).
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Current, nationally representative data on rotavirus disease burden and rotavirus strains in India are needed to understand the potential health benefits of rotavirus vaccination. The Indian Rotavirus Strain Surveillance Network was established with 4 laboratories and 10 hospitals in 7 different regions of India. At each hospital, children aged <5 years who presented with acute gastroenteritis and required hospitalization with rehydration for at least 6 h were enrolled. A fecal specimen was obtained and was tested for rotavirus with use of a commercial enzyme immunoassay, and strains were characterized using reverse-transcription polymerase chain reaction. From December 2005 through November 2007, rotavirus was found in approximately 39% of 4243 enrolled patients. Rotavirus was markedly seasonal in northern temperate locations but was less seasonal in southern locations with a tropical climate. Rotavirus detection rates were greatest among children aged 6-23 months, and 13.3% of rotavirus infections involved children aged <6 months. The most common types of strains were G2P[4] (25.7% of strains), G1P[8] (22.1%), and G9P[8] (8.5%); G12 strains were seen in combination with types P[4], P[6], and P[8] and together comprised 6.5% of strains. These data highlight the need for development and implementation of effective prophylactic measures, such as vaccines, to prevent the large burden of rotavirus disease among Indian children.
Article
The burden of rotavirus gastroenteritis is greatest in India and other developing countries. With the availability of two licensed vaccines and a number of additional vaccines in various stages of development and trial, analysis of detailed clinical information is essential for the development of a uniform method of severity assessment. Diarrhoeal stool samples from 1001 children <5 years of age hospitalized with gastroenteritis were screened for rotavirus using a commercial enzyme immunoassay. Positive samples were confirmed by genotyping using hemi nested multiplex RT-PCR. Detailed clinical data was collected for gastroenteritis assessment for 934 children and extraintestinal presentations were analyzed in 470 children. Severity scoring was carried out for all children using the Vesikari score and in a subset by Clark's scoring system. Rotavirus was detected in 35.4% of samples tested between December 2005 and November 2008. Clark's and Vesikari scores showed moderate correlation but varied greatly in the categorization of severe disease. Using Clark's scoring, only 1.6% were categorized as presenting with severe disease in comparison to 66.1% by the Vesikari score. Association of extraintestinal symptoms with rotavirus gastroenteritis was not documented in this study. The assessment of disease severity using two common severity scoring systems highlights the difference in the categorization of "severe" disease. This underscores the need for a robust scoring system which is needed for vaccine trial and in post-licensure surveillance, because vaccine efficacy is estimated for protection against severe rotavirus gastroenteritis.
Article
We recently reported that the culture-adapted neonatal rotavirus strain 116E represented the first P type 11 human rotavirus, based on the close relationship of its VP4 protein to that of the bovine serotype G10P11 strain B223. In this study, we demonstrated by sequence analysis and cross-neutralization studies that the VP7 protein of 116E is closely related to those of the human serotype G9 strains. F45 and WI61, but distinct from B223 and other rotaviruses. Low-level cross-neutralization was also observed between strains 116E and B223, probably because of the antigenic similarity of their VP4 proteins. We have demonstrated by RNA-RNA hybridization that strain 116E is a reassortant between strains from the Wa and bovine (KK3-like) genogroups, deriving at least seven genes from the former and at least one gene from the latter. Together with the recent identification of serotype G10P11 newborn rotavirus strains in Bangalore, India (M. Das et al., Virology, 194, 374-379, 1993), these results are consistent with the hypothesis that reassortment may be an important mechanism for generation of rotavirus strains of newborns.
Chapter
This chapter describes terminal glycan structures that are common to different classes of glycans. They are the variable portions of N-glycans, O-glycans, and glycolipids that are attached to the core sugars characteristic of each glycan class. The core structures of N-glycans, O-glycans, and glycolipids (Figure 13.1) are the product of biosynthetic pathways discussed in Chapters 8, 9, and 10. More complicated cores can result from tissue- or cell-type-specific pathways and lead to further structural diversification of glycan chains. The glycan units formed by subultimate and terminal sugars at “outer” positions of a glycan often determine the function(s) or recognition properties of a glycoconjugate.
Article
As new rotavirus vaccines are being introduced in immunization programs, global and national estimates of disease burden, especially rotavirus-associated mortality, are needed to assess the potential health benefits of vaccination and to monitor vaccine impact. We identified 76 studies that were initiated after 1990, lasted at least 1 full year, and examined rotavirus among >100 children hospitalized with diarrhea. The studies were assigned to 5 groups (A-E) with use of World Health Organization classification of countries by child mortality and geography. For each group, the mean rotavirus detection rate was multiplied by diarrhea-related mortality figures from 2004 for countries in that group to yield estimates of rotavirus-associated mortality. Overall, rotavirus accounted for 527,000 deaths (95% confidence interval, 475,000-580,000 deaths) annually or 29% of all deaths due to diarrhea among children <5 years of age. Twenty-three percent of deaths due to rotavirus disease occurred in India, and 6 countries (India, Nigeria, Congo, Ethiopia, China, and Pakistan) accounted for more than one-half of deaths due to rotavirus disease. The high mortality associated with rotavirus disease underscores the need for targeted interventions, such as vaccines. To realize the full life-saving potential of vaccines, it will be vital to ensure that they reach children in countries with high mortality. These baseline figures will allow future assessment of vaccine impact on rotavirus-associated mortality.
Article
Rotavirus vaccine has proved effective for prevention of severe rotavirus gastroenteritis in infants in developed countries, but no efficacy studies have been done in developing countries in Asia. We assessed the clinical efficacy of live oral pentavalent rotavirus vaccine for prevention of severe rotavirus gastroenteritis in infants in Bangladesh and Vietnam. In this multicentre, double-blind, placebo-controlled trial, undertaken in rural Matlab, Bangladesh, and urban and periurban Nha Trang, Vietnam, infants aged 4-12 weeks without symptoms of gastrointestinal disorders were randomly assigned (1:1) to receive three oral doses of pentavalent rotavirus vaccine 2 mL or placebo at around 6 weeks, 10 weeks, and 14 weeks of age, in conjunction with routine infant vaccines including oral poliovirus vaccine. Randomisation was done by computer-generated randomisation sequence in blocks of six. Episodes of gastroenteritis in infants who presented to study medical facilities were reported by clinical staff and from parent recollection. The primary endpoint was severe rotavirus gastroenteritis (Vesikari score >or=11) arising 14 days or more after the third dose of placebo or vaccine to end of study (March 31, 2009; around 21 months of age). Analysis was per protocol; infants who received scheduled doses of vaccine or placebo without intervening laboratory-confirmed naturally occurring rotavirus disease earlier than 14 days after the third dose and had complete clinical and laboratory results were included in the analysis. This study is registered with ClinicalTrials.gov, number NCT00362648. 2036 infants were randomly assigned to receive pentavalent rotavirus vaccine (n=1018) or placebo (n=1018). 991 infants assigned to pentavalent rotavirus vaccine and 978 assigned to placebo were included in the per-protocol analysis. Median follow up from 14 days after the third dose of placebo or vaccine until final disposition was 498 days (IQR 480-575). 38 cases of severe rotavirus gastroenteritis (Vesikari score >or=11) were reported during more than 1197 person-years of follow up in the vaccine group, compared with 71 cases in more than 1156 person years in the placebo group, resulting in a vaccine efficacy of 48.3% (95% CI 22.3-66.1) against severe disease (p=0.0005 for efficacy >0%) during nearly 2 years of follow-up. 25 (2.5%) of 1017 infants assigned to receive vaccine and 20 (2.0%) of 1018 assigned to receive placebo had a serious adverse event within 14 days of any dose. The most frequent serious adverse event was pneumonia (vaccine 12 [1.2%]; placebo 15 [1.5%]). In infants in developing countries in Asia, pentavalent rotavirus vaccine is safe and efficacious against severe rotavirus gastroenteritis, and our results support expanded WHO recommendations to promote its global use. PATH (GAVI Alliance grant) and Merck.