JOURNAL OF CLINICAL MICROBIOLOGY, Aug. 2007, p. 2641–2648
Copyright © 2007, American Society for Microbiology. All Rights Reserved.
Vol. 45, No. 8
Development and Validation of DNA Microarray for Genotyping
Group A Rotavirus VP4 (P, P, P, P, and P)
and VP7 (G1 to G6, G8 to G10, and G12) Genes?†
Shinjiro Honma,1‡ Vladimir Chizhikov,2Norma Santos,1,3Masatoshi Tatsumi,1
Maria do Carmo S. T. Timenetsky,4Alexandre C. Linhares,5
Joana D’Arc P. Mascarenhas,5Hiroshi Ushijima,6
George E. Armah,7Jon R. Gentsch,8
and Yasutaka Hoshino1*
Epidemiology Section, Laboratory of Infectious Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of
Health, Bethesda, Maryland1; Laboratory of Method Development, Center for Biologics Evaluation and Research, Food and
Drug Administration, Rockville, Maryland2; Instituto de Microbiologia, Universidade Federal do Rio de Janeiro, Rio de Janerio,
Brazil3; Instituto Adolfo Lutz, Sa ˜o Paulo, Brazil4; Instituto Evandro Chagas, Secretaria de Vigila ˆncia em Sau ´de, Bele ´m,
Brazil5; University of Tokyo, Tokyo, Japan6; University of Ghana, Legon, Ghana7; and Gastroenteritis and
Respiratory Viruses Laboratory Branch, Centers for Disease Control and Prevention, Atlanta, Georgia8
Received 4 April 2007/Returned for modification 14 May 2007/Accepted 4 June 2007
Previously, we reported the development of a microarray-based method for the identification of five clinically
relevant G genotypes (G1 to G4 and G9) (V. Chizhikov et al., J. Clin. Microbiol. 40:2398–2407, 2002). The
expanded version of the rotavirus microarray assay presented herein is capable of identifying (i) five clinically
relevant human rotavirus VP4 genotypes (P, P, P, P, and P) and (ii) five additional human
rotavirus VP7 genotypes (G5, G6, G8, G10, and G12) on one chip. Initially, a total of 80 cell culture-adapted
human and animal reference rotavirus strains of known P (P to P, P, P, and P) and G
(G1-6, G8 to G12, and G14) genotypes isolated in various parts of the world were employed to evaluate the new
microarray assay. All rotavirus strains bearing P, P, P, P, or P and/or G1 to G6, G8 to G10,
or G12 specificity were identified correctly. In addition, cross-reactivity to viruses of genotype G11, G13, or G14
or P to P, P, P, P to P, P, or P was not observed. Next, we analyzed a total of 128
rotavirus-positive human stool samples collected in three countries (Brazil, Ghana, and the United States) by
this assay and validated its usefulness. The results of this study showed that the assay was sensitive and specific
and capable of unambiguously discriminating mixed rotavirus infections from nonspecific cross-reactivity; the
inability to discriminate mixed infections from nonspecific cross-reactivity is one of the inherent shortcomings
of traditional multiplex reverse transcription-PCR genotyping. Moreover, because the hybridization patterns
exhibited by rotavirus strains of different genotypes can vary, this method may be ideal for analyzing the
genetic polymorphisms of the VP7 or VP4 genes of rotaviruses.
Group A rotaviruses remain the single most important eti-
ologic agents of severe diarrhea in infants and young children
worldwide. Rotavirus diarrhea has been estimated to be re-
sponsible for a median of 611,000 deaths annually in children
under 5 years of age, predominantly in developing countries
(43). In the United States, approximately 2.7 million children
are affected by rotavirus illness each year, resulting in about 20
deaths, 50,000 hospitalizations, 500,000 physician visits, and
more than 1 billion U.S. dollars in societal costs (5, 19, 20, 30,
43). Because of the significant morbidity and mortality associ-
ated with rotavirus diarrhea, the development and implemen-
tation of a safe and effective rotavirus vaccine has been an
important public health priority. Recently, two rotavirus vac-
cines have been licensed in many countries; the effectiveness of
such vaccines in poor populations in certain sub-Saharan
African as well as Southeast Asian countries remains to be
Rotaviruses are nonenveloped, icosahedral viruses of the
family Reoviridae with 11 genomic segments of double-
stranded RNA, each encoding at least one structural or non-
structural protein. The rotavirus outer capsid proteins VP7
(which defines G types) and VP4 (which defines P types) evoke
neutralizing antibodies independently (22, 25, 42). In addition,
since neutralizing antibodies appear to play an important role
in protection against rotavirus disease and infection in a sero-
type-specific manner (for reviews, see references 26 and 29),
rotavirus strain surveillance (i.e., G and/or P type determina-
tion) has been conducted throughout the world. Various assays
have been developed to determine G and P serotypes of rota-
viruses, including type-specific-monoclonal antibody (MAb)-
based enzyme-linked immunosorbent assay (ELISA) (2, 10, 55,
57) and a neutralization assay using type-specific polyclonal
antibodies (18, 26, 60). However, because of the lack of ap-
* Corresponding author. Mailing address: National Institutes of
Health, Building 50, Room 6308, 50 South Drive, MSC 8026,
Bethesda, MD 20892-8026. Phone: (301) 594-1851. Fax: (301) 480-
1387. E-mail: firstname.lastname@example.org.
† Supplemental material for this article may be found at http://jcm
‡ Present address: Department of Pediatrics, Sapporo University
School of Medicine, Sapporo, Japan.
?Published ahead of print on 13 June 2007.
propriate and readily available reagents (e.g., G or P type-
specific high-titer polyclonal antisera; MAb to G5, G6, G8, G9,
or G10; and MAbs to various P types), serotyping is not an
available option for processing a variety of rotavirus field sam-
ples. Genotyping by reverse transcription PCR (RT-PCR) de-
veloped in the early 1990s as a proxy method for serotyping,
which employs a set of genotype-specific primers (multiplex
RT-PCR), is rapid, simple, and sensitive (11, 14, 15, 21, 24, 58).
Today, multiplex RT-PCR is the most widely used method for
the identification of rotavirus G and P genotypes. However,
there are several drawbacks in this method; for example, (i)
because a majority of genotype-specific primers used in mul-
tiplex RT-PCR are designed from the sequences of strains
isolated more than a decade ago, they may carry sequence
mismatches in the primer binding regions of the target gene(s)
of more-recent rotavirus strains, resulting in decreased sensi-
tivity, and (ii) because a genotype is determined only by the
size of PCR products in a gel, it is not uncommon to encounter
a situation in which the possibility that certain spurious bands
are present cannot be excluded or a mixed infection cannot be
incriminated. Indeed, it has been reported that genotyping
results could vary depending upon the primer pairs used in the
multiplex RT-PCR due to the accumulation of point mutations
in primer binding regions of the VP4 or VP7 gene (1, 4, 13, 27,
28, 38, 44, 48, 52). Not only for epidemiological rotavirus strain
surveillance but also for effective rotavirus vaccine develop-
ment, there is a need for sensitive and reliable diagnostic
techniques which do not bear such disadvantages. Previously,
we reported the development of a rapid and reliable method
for the identification of clinically relevant human rotavirus G
genotypes (i.e., G1 to G4 and G9) using oligonucleotide mi-
croarray hybridization (8). By using this method, which com-
bines the sensitivity of PCR and the specificity of hybridization,
we were successful in detecting and unambiguously identifying
such G genotypes. Although 16 G genotypes and 28 P geno-
types have been identified thus far (23, 36, 37, 46, 56), five G
genotypes (G1 to G4 and G9) and two P genotypes (P and
P) have been repeatedly shown worldwide to be of epide-
miologic importance in humans (16, 17, 30, 32, 51). More
recently, however, G genotypes other than G1 to G4 and G9
and P genotypes other than P and P have been detected
in various parts of the world and include G5, G6, G8, G10,
G12, P, P, and P (for reviews, see references 17, 47,
and 51). In this study, we modified the original microarray
assay and expanded it to include the identification of (i) five
clinically relevant human rotavirus VP4 genotypes (P, P,
P, P, and P) and (ii) five additional human rotavirus
VP7 genotypes (G5, G6, G8, G10, and G12). The usefulness
and validity of this assay were confirmed by analyzing a total of
128 stool rotaviruses collected between 1990 and 2004 from
three countries. In addition, we evaluated whether this method
could be applied to analyze the genetic polymorphism of the
VP7 or VP4 genes of rotaviruses in a given G or P genotype
isolated in different parts of the world.
MATERIALS AND METHODS
Rotavirus strains. A total of 80 cell culture-adapted human and animal ref-
erence rotavirus strains of known G and P genotypes were tested in this study
(see Table S1 in the supplemental material). These strains, which were isolated
from humans and eight different animal species in 19 countries on five continents
between 1958 and 1999, represented collectively 13 G genotypes (G1 to G6 and
G8 to G14) and 15 P genotypes (P to P, P, P, P, and P)
(See Table S1 in the supplemental material). Each of the 80 rotavirus strains was
plaque purified three times in MA104 monkey kidney cells prior to use.
Rotavirus-positive human stool samples. For further validation of this assay,
we analyzed a total of 128 rotavirus-positive human stool field samples collected
in three countries (Brazil, Ghana, and the United States) between 1990 and 2004,
which were all previously analyzed by RT-PCR.
RNA extraction, RT-PCR, and nested PCR. Viral double-stranded RNA was
extracted with TRIzol (Invitrogen) from infected cell culture lysates or stool
suspensions (approximately 10%) as described previously (53). In order to in-
crease the sensitivity of the microarray assay, gene amplifications were carried
out in a first-round RT-PCR, followed by a nested PCR. RT was performed as
follows: extracted double-stranded RNA (1 ?l) was added to 10 ?l of 1?
first-strand buffer containing 10 mM Tris-HCl (pH 8.3); 40 mM KCl; 1.5 mM
MgCl2; a 200 ?M concentration (each) of dATP, dCTP, dGTP, and dTTP; 5 mM
dithiothreitol; 3.5% dimethyl sulfoxide; and a 100 pM concentration (each) of
two primers (Beg9 and End9 for VP7 and F1 and C10 for VP4) listed in Table
S2 in the supplemental material. The mixture was heated at 97°C for 5 min and
placed on ice for 5 min, and then 2 U of SuperScript II (Invitrogen) was added.
The tubes were set in a GeneAmp PCR system thermocycler (Applied Biosys-
tems) and incubated at 42°C for 45 min. First-round PCR was performed in 25
?l of 1? storage buffer B containing 2.5 mM MgCl2, a 200 nM concentration of
each primer, a 100 ?M concentration of each of the four deoxynucleoside
triphosphates, 5 U of Taq DNA polymerase (Promega), and 1 ?l of the RT
product. Nested PCR was carried out in 25 ?l of the same reaction mixture
except that the different primer sets (LID3 and G922 for VP7 and F4 and C8 for
VP4) and the first-round-PCR product (1 ?l) were included. In both PCR steps,
amplification was done under the following conditions: initial denaturation at
94°C for 2 min; 30 cycles at 94°C for 30 s, 60°C for 30 s, and 72°C for 2 min; and
a final extension at 72°C 5 min. The PCR products were analyzed by electro-
phoresis in 1% agarose gel in 1? Tris-acetate-EDTA buffer containing 0.2 ?g/ml
of ethidium bromide.
Synthesis of Cy5-labeled DNA samples. The fluorescently labeled single-
stranded DNA samples for hybridization were generated by the single primer
extension (PE) reaction in the presence of a single forward primer used in the
nested PCR. The reaction was performed in 25 ?l of a reaction mixture con-
taining 1? storage buffer B with 2.5 mM MgCl2; a 600 nM concentration of each
forward primer; a 200 nM concentration (each) of dGTP, dATP, and dTTP; 40
nM dCTP; 40 nM Cy5-dCTP; 5 U of Taq DNA polymerase (Promega); and 4 to
10 ?l of a DNA template of the purified (QIAquick PCR purification kit)
nested-PCR product. The following conditions were used in the PE reaction: 1
min at 94°C; 40 cycles of 30 s at 94°C, 45 s at 55°C, and 2 min at 72°C; and a final
intubation for 10 min at 72°C. Cy5-labeled samples were separated from nonin-
corporated Cy5-dCTP by centrifugation on Centri-Sep columns (Princeton Sep-
Synthesis of a Cy3-labeled QC oligonucleotide. The Cy3-labeled quality con-
trol (QC) oligonucleotide was prepared as described previously (8).
Design of genotype-specific oligonucleotide probes. More than 720 VP7 gene
sequences and 450 VP4/VP8* gene sequences obtained from GenBank were
aligned and analyzed with MacVecor 6.0 (Accelrys). Sequences of highly con-
served regions within a given G or P genotype were selected to design genotype-
specific oligonucleotide probes which are listed in Table S2 in the supplemental
material. The 5? end of each probe bore an amino-link group to immobilize the
probe effectively on silylated (aldehyde-coated) glass slides (CEL Associates,
Microchip fabrication. Microchip fabrication was performed as previously
described (8), with a slight modification. Microarray chips were printed using a
contact microspotting robot (Cartesian Technologies, Inc.) and a ChipMaker
microspotting device (TeleChem International, Inc.). The average spot diameter
was 250 ?m. The final spotting solution contained a 100 ?M concentration of a
genotype-specific oligonucleotide probe and a 20 ?M concentration of a QC
oligonucleotide in 0.25 M acetic acid. After the chips were printed, slides were
dehydrated for 15 min at 80°C and incubated for 10 min in a freshly prepared
0.25% solution of NaBH4. Then the slides were washed once for 5 min with 0.2%
sodium dodecyl sulfate in water and five times for 1 min each with distilled water.
Hybridization. Hybridization was performed at 45°C for 30 min in an incuba-
tion chamber (ArrayIt). Immediately before hybridization, 1.8 ?l of the Cy5-
labeled sample was mixed with an equal volume of 2? hybridization buffer
containing the QC oligonucleotide labeled with 0.15 ?M Cy3. The mixture was
heated at 99°C for 1 min to denature double-stranded DNA, followed by chilling
on ice. The hybridization aliquot was applied to the array chip and covered with
a plastic coverslip (4 by 7 mm) to prevent evaporation of the sample during
2642HONMA ET AL. J. CLIN. MICROBIOL.
incubation. After hybridization, the slide was washed once for 1 min with 6? SSC
(1? SSC is 0.15 M NaCl plus 0.015 sodium citrate) containing 0.2% Tween 20,
once for 2 min with 6? SSC, and once for 2 min with 2? SSC and then dried by
an air stream.
Microarray scanning and data analysis. Fluorescent images of the microchip
were generated by scanning the slides by using a confocal GenePix 4100A
personal fluorescence scanner (Axon Instruments). The fluorescent signals from
each spot obtained at 570 nm (Cy3) and 694 nm (Cy5) were analyzed using
GenePix Pro 5.0 software (Axon). Background signals obtained from the region
surrounding each spot were subtracted, and the resulting absolute value of the
Cy5 fluorescent signal from each probe was divided by the Cy3 signal from the
QC probe of the same spot. Fluorescent signals with a statistically significant
difference (P ? 0.01) from the average background level were considered to be
RT-PCR. A total of 80 rotavirus samples each of which bore
one of 13 G genotypes (G1 to G6 and G8 to G14) and one of
15 P genotypes (P to P, P, P, and P) were
analyzed by first-round and nested PCR. For first-round PCR,
we used a conventional primer pair, Beg9 and End 9 (21), to
synthesize full-length VP7 genes (1,062 bp). Seventy-five of the
80 (93.8%) samples were positive by first-round PCR. In order
to increase the sensitivity, we carried out nested PCR on the
first-round-PCR products using the primer pair LID3 and 922
(see Table S2 in the supplemental material) and successfully
amplified all 80 samples. The size of amplicons generated by
nested PCR was 894 bp. For the amplification of the VP4
genes, we designed four new PCR primers (F1, F4, C8, and
C10) from conserved regions of the VP4 gene. Two conserved
regions, from nucleotides 1 to 20 and 4 to 23, were used to
design the forward primers F1 and F4, respectively, and two
other conserved regions, from nucleotides 1446 to 1467 and
1595 to 1618, were selected to make the reverse primers C8
and C10, respectively (Table S2 in the supplemental material).
The sensitivity of the first-round PCR using the F1 and C10
primer pair was 91.3% (73 of 80 samples). All 80 strains used
in this study were amplified by nested PCR using the F4 and C8
primer pair. The sizes of amplicons generated by the first-
round and nested PCRs were 1,618 bp and 1,464 bp, respec-
Sensitivity of the oligonucleotide microarray assay. To de-
termine the sensitivity of the oligonucleotide microarray assay,
the nested-PCR products of the VP4 or VP7 gene of human
rotavirus strain D (PG1) were analyzed. Figure 1A shows a
gel electrophoresis analysis of serially diluted (fivefold) cDNA
amplicons of the VP7 (lanes 2 to 6) and VP4 (lanes 7 to 11)
genes of strain D. The amount of cDNA in the initial reaction
was 1 ?g/?l (lanes 2 and 7); this was followed by a fivefold
dilution series (lanes 2 to 6 and 8 to 11). Each diluted sample
was purified and used as a template for the PE reaction under
the conditions described in Materials and Methods. The
scanned fluorescent images and fluorescence intensities of
these samples are shown in Fig. 1B and C, respectively. The
minimum detectable concentration of the nested-PCR product
was 8 ng/?l, which corresponded to the minimum concentra-
tion that produced a visible band in a 1% agarose gel after
Design and fabrication of G and P genotyping array chip.
We previously reported the design and fabrication of an oli-
gonucleotide microarray chip for the identification of five clin-
ically relevant G genotypes (G1 to G4 and G9) of human
rotaviruses (8). We recently designed genotype-specific oligo-
nucleotide probes for five additional human rotavirus G geno-
types (G5, G6, G8, G10, and G12). After eliminating cross-
reactive probes, we selected seven probes for each G genotype.
FIG. 1. Sensitivity of the oligonucleotide microarray hybridization
assay. (A) Gel electrophoresis analysis of serially fivefold-diluted nest-
ed-PCR products of the VP7 gene (lanes 2 to 6) and VP4 gene (lanes
7 to 11) of human rotavirus D strain (G1,P). A 10-?l sample of
cDNA amplicons of each concentration was electrophoresed in a 0.8%
agarose gel and stained with ethidium bromide. Lanes: 1 and 12, 1-kb
DNA ladder mix; 2 and 7, 1 ?M; 3 and 8, 200 nM; 4 and 9, 40 nM; 5
and 10, 8 nM; and 6 and 11, 1.6 nM. (B) Scanned images of fluores-
cently labeled cDNA samples of the VP7 or VP4 gene of the D strain
hybridized to a microchip. Cy5-labeled samples of the VP7 and VP4
genes generated by PE were mixed immediately before hybridization.
The concentration of cDNA amplicons used for PE were as follows:
panel 1, 1 ?M; panel 2, 200 nM; panel 3, 40 nM; panel 4, 8 nM; panel
5, 1.6 nM; and panel 6, 0 nM (distilled water). (C) Fluorescence
intensities of the seven genotype G1-specific probes scanned from
images in panel B. Numbers 1 to 6 in panel C correspond to images 1
to 6 in panel B.
VOL. 45, 2007 GENOTYPING OF ROTAVIRUSES BY MICROARRAY ANALYSIS2643
For P genotyping, we designed 9 to 12 genotype-specific oli-
gonucleotide probes for each of five clinically relevant human
rotavirus P genotypes (i.e., P, P, P, P, and P)
(see Table S1 in the supplemental material). Oligonucleotide
probes complementary to the primers used for nested PCR
served as positive controls. The sequences of anti-LID3 and
anti-F4 were complementary to those of LID3 and F4, respec-
tively (Table S2 in the supplemental material). Each genotype-
specific probe and positive-control probe were mixed with
QCprb (an oligonucleotide complementary to the Cy3-labeled
QC probe) before the chips were printed onto glass slides.
Thus, each spot on the glass slide contained QCprb not only
for an evaluation of spotting reproducibility and hybridization
efficiency but also for normalization of data. Figure 2A shows
the newly designed rotavirus G and P genotyping microarray
Analysis of fluorescent images. By analyzing scanned fluo-
rescent images of microchips, we could detect and discriminate
successfully all 78 samples belonging to one of 10 G genotypes
(G1 to G6, G8 to G10, and G12) and all 52 samples bearing
one of 5 P genotypes (P, P, P, P, and P) (see
Table S1 in the supplemental material). In addition, no signif-
icant cross-reactivity was detected from 3 strains belonging to
the G11, G13, or G14 genotype and 28 strains belonging to the
P to P, P, P, P, P, P, P, or P
genotype, against which no specific probes were available on
the chip. Figure 2B shows fluorescent images of microchips
obtained from six rotavirus strains bearing one of five G (G2,
G5, G6, G10, and G12) and one of five P (P, P, P,
P, and P) genotypes. In general, the specificities of all
genotype-specific probes were high; however, there were a few
cross-reactive probes that hybridized with samples of heterol-
ogous genotypes. For example, (i) probe G2-8 hybridized with
two G10 strains (B223 [not shown] and KC-1 [Fig. 2B]) and (ii)
probes G5-5, G6-4, and G9-5 hybridized with G3 strains Cat2,
HCR3, and RRV, respectively. Among the P genotype-specific
probes, only two probes, P4-1 and P14-6, cross-hybridized with
a P strain, US1205, and a P strain, BD524, respectively.
Of note was the finding that each cross-reactive probe hybrid-
ized with some but not all strains within a given genotype.
Furthermore, there were no samples that hybridized with more
than one heterologous probe, and in addition, the intensities of
all cross-reactive signals were low.
Fluorescence intensity profiles. Figure 3 shows the quanti-
tative fluorescence profiles of selected rotavirus strains bearing
P, P, P, P, or P specificity after analysis with
GenePix Pro 5.0 software. The normalized maximum of quan-
titative fluorescence (Cy5) signals (percentage) of each oligo-
nucleotide probe is shown on the y axis. In such profiles, the
unique hybridization pattern of each rotavirus strain within a
given G or P genotype can be recognized more clearly than in
scanned fluorescent images (Fig. 2B). Because of spontaneous
mutations in the probe binding regions of the VP4 or VP7
gene, different strains within the same genotype tended to
display diverse patterns of hybridization.
Analysis of rotavirus-positive human field stool samples by
microarray. In order to validate the microarray assay, we an-
alyzed a total of 128 rotavirus-positive stool samples; 64 were
from Brazil, 31 from Ghana, and 33 from the United States.
Each of the samples was analyzed previously by RT-PCR in
each country. Tables 1 and 2 summarize the results. Several
interesting features emerged from this study: (i) all the G
and/or P genotypes that were previously nontypeable by RT-
PCR were typed by microarray analysis, and (ii) a relatively
large number of samples gave discordant genotyping results in
the two assays. Because of this, we used the third assay, PCR-
ELISA, and confirmed the microarray results. In the PCR-
ELISA, the biotinylated VP7 or VP4 gene amplicons gener-
ated by RT-PCR were immobilized onto streptavidin-coated
96-well microplates and hybridized to digoxigenin-labeled G or
FIG. 2. (A) Layout of the rotavirus G (yellow) and P (blue) geno-
type-specific probes on the microarray chip. “G” and “P” in orange
circles represent VP7 (anti-LID3) and VP4 (anti-F4) positive-control
probes, respectively. (B) Scanned fluorescent images of hybridization
patterns of six rotavirus strains each bearing various G-P combinations,
as noted. Cy5 fluorescence-positive signals and Cy3 fluorescence QC
signals were merged by using GenePix Pro 5.0 software.
2644 HONMA ET AL.J. CLIN. MICROBIOL.
P type-specific oligoprobes (three probes per type). The hy-
brids were then detected using antidigoxigenin Fab fragment
labeled with peroxidase, and the reaction was measured spec-
trophotometrically (N. Santos et al., unpublished data).
Microarray-based techniques have been well established as
powerful tools in various fields of molecular biology (6, 12, 45,
49). In virology, this method has been used for analyses of gene
expression profiles of cells infected with various viruses. Re-
cently, this assay has been applied for genotyping of selected
viruses, including polioviruses, hepatitis B viruses, influenza
viruses, hantaviruses, coxsackieviruses, papillomaviruses, mea-
sles virus, adenoviruses, flaviviruses, poxviruses, herpesviruses,
and mumps viruses (3, 7–9, 31, 33, 34, 39–41, 50, 54, 59). We
reported previously the successful development and evaluation
of an oligonucleotide microarray hybridization methodology
for the identification of five clinically relevant human rotavirus
G genotypes (i.e., G1 to G4 and G9) (8). More recently, an
increasing number of epidemiological studies have reported
the detection in diarrheal patients of uncommon G types, in-
cluding G5, G6, G8, G10, and G12, in both developing and
developed countries. Thus, in this study, we modified the orig-
inal microarray assay and expanded it to include the identifi-
cation of such unusual G types as well as clinically relevant P
genotypes (i.e., P, P, P, P, and P). Hence, this
microarray assay can now identify almost all G and P geno-
types of human rotaviruses that have been detected thus far.
Epidemiological surveillance of rotavirus G and P types before
and after an introduction of a candidate rotavirus vaccine in
various parts of the world is important in order to evaluate (i)
temporal and geographic changes/fluctuations of rotavirus ge-
notype distribution, (ii) relationships between vaccine efficacy
and circulating rotavirus strains, and (iii) horizontal transmis-
sion of vaccine strains.
The analytical sensitivity of the microarray method reported
in this study is very high. The minimum concentration of
cDNA that could be detected and typed was 8 ng/?l. In gen-
eral, samples that produced a visible band in an ethidium
bromide-stained 1% agarose gel after the first-round or nested
PCR were all successfully P or G genotyped. By applying
nested PCR to the first-round-PCR products of strains that
gave no visible bands (and therefore could not be typed), we
could successfully amplify and genotype both the VP4 and VP7
genes of such rotavirus strains. Thus, because of the high
sensitivity of primer pairs used in the nested PCR in this study,
the combination of first-round and nested PCR was shown to
FIG. 3. Quantitative fluorescence (Cy5) profiles of rotavirus P genotype microarray of representative rotavirus strains belonging to P, P,
P, P, or P. Normalized percent fluorescent signals from each oligoprobe are shown on the y axis. Locations of probes are shown on the
x axis and are marked with different colors: dark blue for P, orange for P, light blue for P, pink for P, and green for P.
VOL. 45, 2007 GENOTYPING OF ROTAVIRUSES BY MICROARRAY ANALYSIS2645
have a potential of amplifying both the VP4 and VP7 genes of
strains bearing not only common but also uncommon G or P
The specificity of this microarray method is also very high.
We could correctly and unambiguously identify both P and G
genotypes of all reference rotavirus strains belonging to geno-
types against which the genotype-specific microarray probes
were designed. In addition, no cross-reactivity with rotavirus G
or P genotypes against which specific probes were unavailable
on the chip was detected. Thus, strains which do not generate
fluorescent signals can safely be considered to bear a G geno-
type other than G1 to G6, G8 to G10, and G12 and/or a P
genotype other than P, P, P, P, and P.
Only four G and two P genotype-specific probes were found
to be cross-reactive with samples of heterologous genotypes.
Of note is the finding, however, that each of the samples that
exhibited cross-reactivity reacted with only one heterologous
probe. Moreover, the intensity of all cross-reactive signals was
low. It has been well recognized that it is necessary to distin-
guish mixed rotavirus infections from cross-reactivity in multi-
plex RT-PCRs (14) by using a confirmatory assay for the indi-
vidual genotypes. In contrast to PCR, the microarray method
has the inherent ability to readily identify individual genotypes
for the mixed infections. In such a case, unlike with cross-
reactive signals, more than one probe of suspected G and/or P
genotypes would produce strong positive fluorescence signals.
Moreover, in this microarray assay, such cross-reactive signals
can be utilized for accurate discrimination among various
strains within the same genotype. For example, bovine G10
rotavirus KC-1 and B223 strains reacted with the heterologous
G2-7 probe with low intensities, while human G10 rotavirus
A64 strain exhibited no reactivity with the same probe.
While this study was in progress, Lovmar et al. reported (35)
a genotyping method of five G (G1 to G4 and G9) and four P
(P, P, P, and P) types of rotavirus by using PE in a
microarray format. Their method appears to differ from ours in
various aspects. First, because discrimination of genotypes de-
pends on the difference in the 3? ends between genotype-
specific probes and the target genome segments of samples,
this method may not work well with strains that have a muta-
tion in this region. For example, among the strains tested in the
present study, (i) two G3 strains (HCR3 and Ro1843) which
have 3? ends different from that of each of three G3-specific
probes (G3-1, G3-2, and G3-3) reported by Lovmar et al. could
not be typed (false-negative result), and (ii) three or four
sequence mismatches found between two G4 strains (Gottfried
and SB1A) and the three G4-specific probes (G4-1, G4-2, and
G4-3) described by Lovmar and coworkers may lead to a wrong
conclusion (false-negative result). Second, in their assay, it may
be difficult in some cases to distinguish mixed rotavirus infec-
tions from cross-reactions that may cause a false-positive re-
sult. For example, because there is only one base mismatch
between the G10 strains (B223 and KC-1) tested in the present
study and one of their G2-specific probes (G2-6) and because,
in addition, 3? ends are complementary to each other, such
G10 samples may be misdiagnosed as a mixture of G10 and G2
viruses (false-positive result). Third, this method can provide
only limited information about 3?-end differences among
strains within the same genotype. Previously, we reported that
our microarray method was capable of “subgenotyping” viruses
in a given G genotype (G1 to G4 and G9) (8). In the present
study, not only the VP7 gene but also the VP4 gene of each
rotavirus strain was found to display a unique hybridization
pattern that correlated closely with its nucleotide sequences.
Thus, one of the major advantages of our new microarray
TABLE 1. G and P genotypes of stool rotavirus samples
determined by RT-PCR and microarray assays
Location (yr) where stool
samples were collected
Genotype(s) determined by
Brazil (1990–2000) 14
United States (1997–2000)32 G1
aDiscrepant G and/or P types are in boldface. NT, nontypeable.
TABLE 2. Concordance and discordance in genotyping results determined by RT-PCR and microarray assays
Origin of samples (n)
No. (%) of G genotypes that were:No. (%) of P genotypes that were:
United States (33)
aResults were cocordant between RT-PCR and microarray analysis.
bResults were discordant between RT-PCR and microarray analysis.
cStrains were nontypeable by RT-PCR but typeable by microarray analysis.
2646 HONMA ET AL.J. CLIN. MICROBIOL.
method is that it is ideal for analyzing such genetic polymor-
phisms of the VP7 and VP4 genes of various rotavirus strains
within the same genotype. In addition, we found that the ex-
istence of even one nucleotide mismatch located near the 3? or
5? end of a probe changed its pattern of hybridization profile
(data not shown). In general, rotavirus strains in a given ge-
notype that are isolated (i) at different locations, (ii) in differ-
ent years even at the same location, or (iii) from different
animal species display different hybridization profiles. For ex-
ample, two Japanese G2 strains, KUN and S2, displayed sim-
ilar hybridization patterns which were distinct from that dis-
played by U.S. G2 strain DS-1 or Venezuelan G2 strain HN126
(data not shown). These results indicate that this assay can be
applied to monitoring the evolutionary divergence and genetic
drift of the selected rotavirus genes, including the VP7 and
VP4 genes. Although sequencing of the target genome seg-
ment may be needed for detailed analysis, it is time-consuming,
expensive, and not practical for analyses of a large number of
field isolates. This microarray method is ideal for a rapid
screening of a large number of rotavirus strains to detect and
analyze genetic polymorphism before sequencing. Analyses by
this assay of a possible genetic/antigenic drift of the VP7
and/or VP4 gene of G1P viruses collected longitudinally in
three countries (Brazil, Spain, and the United States) are un-
der way in this laboratory.
We thank Michael Wilson (Microarray Facility Section, Research
Technologies Branch, National Institute of Allergy and Infectious Dis-
eases, National Institutes of Health) for the assistance in preparation
of microchips. We also thank G. N. Gerna, M. E. Thouless, K. Banyai,
G. Szucs, R. A. Hesse, and D. R. Snodgrass for kindly providing us
with various rotavirus stains.
This research was supported in part by the Intramural Research
Program of the National Institute of Allergy and Infectious Disease,
National Institutes of Health.
There is no conflict of interest to declare.
1. Adah, M. I., A. Rohwedder, O. D. Olaleyle, and H. Werchau. 1997. Nigerian
rotavirus serotype G8 could not be typed by PCR due to nucleotide mutation
at the 3? end of the primer binding site. Arch. Virol. 142:1881–1887.
2. Akatani, K., and N. Ikegami. 1987. Typing of fecal rotavirus specimens by
enzyme-linked immunosorbent assay using monoclonal antibodies. Clin.
Virol. 15:61–68. (In Japanese.)
3. Amexis, G., S. Rubin, V. Chizhikov, F. Pelloquin, K. Carbone, and K. Chu-
makov. 2002. Sequence diversity of Jeryl Lynn strain of mumps virus: quan-
titative mutant analysis for vaccine quality control. Virology 300:171–179.
4. Banyai, K., J. R. Gentsch, R. Schipp, F. Jakab, E. Meleg, I. Mihaly, and G.
Szucs. 2005. Dominating prevalence of P, G1 and P, G9 rotavirus
strains among children admitted to hospital between 2000 and 2003 in
Budapest, Hungary. J. Med. Virol. 76:414–423.
5. Bresee, J. S., R. I. Glass, B. Ivanoff, and J. R. Gentsch. 1999. Current status
and future priorities for rotavirus vaccine development, evaluation and im-
plementation in developing countries. Vaccine 17:2207–2222.
6. Bryant, P. A., D. Venter, R. Robins-Browne, and N. Curtis. 2004. Chips with
everything: DNA microarrays in infectious diseases. Lancet Infect. Dis.
7. Cherkasova, E., M. Laassri, V. Chizhikov, E. Korotkova, E. Dragunsky, V. I.
Agol, and K. Chumakov. 2003. Microarray analysis of evolution of RNA
viruses: evidence of circulation of virulent highly divergent vaccine-derived
polioviruses. Proc. Natl. Acad. Sci. USA 100:9398–9403.
8. Chizhikov, V., M. Wagner, A. Ivshina, Y. Hoshino, A. Z. Kapikian, and K.
Chumakov. 2002. Detection and genotyping of human group A rotaviruses
by oligonucleotide microarray hybridization. J. Clin. Microbiol. 40:2398–
9. Choi, B. S., O. Kim, M. S. Park, K. S. Kim, J. K. Jeong, and J. S. Lee. 2003.
Genital human papillomavirus genotyping by HPV oligonucleotide micro-
array in Korean commercial sex workers. J. Med. Virol. 71:440–445.
10. Coulson, B. S., L. E. Unicomb, G. A. Pitson, and R. F. Bishop. 1987. Simple
and specific enzyme immunoassay using monoclonal antibodies for serotyp-
ing human rotaviruses. J. Clin. Microbiol. 25:509–515.
11. Das, B. K., J. R. Gentsch, H. G. Cicirello, P. A. Woods, A. Gupta, M.
Ramachandran, R. Kumar, M. K. Bhan, and R. I. Glass. 1994. Character-
ization of rotavirus strains from newborns in New Delhi, India. J. Clin.
12. Dhiman, N., R. Bonilla, D. J. O’Kane, and G. A. Poland. 2001. Gene ex-
pression microarrays: a 21st century tool for directed vaccine design. Vaccine
13. Fischer, T. K., N. A. Page, D. D. Griffin, J. Eugen-Olsen, A. G. Pedersen, P.
Valentiner-Branth, K. Molbak, H. Sommerfelt, and N. M. Nielsen. 2003.
Characterization of incompletely typed rotavirus strains from Guinea-
Bissau: identification of G8 and G9 types and a high frequency of mixed
infections. Virology 311:125–133.
14. Fischer, T. K., and J. R. Gentsch. 2004. Rotavirus typing methods and
algorithms. Rev. Med. Virol. 14:71–82.
15. Gentsch, J. R., R. I. Glass, P. Woods, V. Gouvea, M. Gorziglia, J. Flores,
B. K. Das, and M. K. Bhan. 1992. Identification of group A rotavirus gene 4
types by polymerase chain reaction. J. Clin. Microbiol. 30:1365–1373.
16. Gentsch, J. R., P. A. Woods, M. Ramachandran, B. K. Das, J. P. Leite, A.
Alfieri, R. Kumar, M. K. Bhan, and R. I. Glass. 1996. Review of G and P
typing results from a global collection of rotavirus strains: implications for
vaccine development. J. Infect. Dis. 174(Suppl. 1):S30–S36.
17. Gentsch, J. R., A. R. Laird, B. Bielfelt, D. D. Griffin, K. Banyai, M. Ram-
achandran, V. Jain, N. A. Cunliffe, O. Nakagomi, C. D. Kirkwood, T. K.
Fischer, U. D. Parashar, J. S. Bresee, B. Jiang, and R. I. Glass. 2005.
Serotype diversity and reassortment between human and animal rotavirus
strains: implications for rotavirus vaccine programs. J. Infect. Dis. 192(Suppl.
18. Gerna, G., N. Passarani, M. Battaglia, and E. Percivalle. 1984. Rapid sero-
typing of human rotavirus strains by solid-phase immune electron micros-
copy. J. Clin. Microbiol. 19:273–278.
19. Glass, R. I., J. Gentsch, and J. C. Smith. 1994. Rotavirus vaccines: success by
reassortment? Science 265:1389–1391.
20. Glass, R. I., U. D. Parashar, J. S. Bresee, R. Turcios, T. K. Fischer, M. A.
Widdowson, B. Jiang, and J. R. Gentsch. 2006. Rotavirus vaccines: current
prospects and future challenges. Lancet 368:323–332.
21. Gouvea, V., R. I. Glass, P. Woods, K. Taniguchi, H. F. Clark, B. Forrester,
and Z. Y. Fang. 1990. Polymerase chain reaction amplification and typing of
rotavirus nucleic acid from stool specimens. J. Clin. Microbiol. 28:276–282.
22. Greenberg, H. B., J. Valdesuso, K. van Wyke, K. Midthun, M. Walsh, V.
McAuliffe, R. G. Wyatt, A. R. Kalica, J. Flores, and Y. Hoshino. 1983.
Production and preliminary characterization of monoclonal antibodies di-
rected at two surface proteins of rhesus rotavirus. J. Virol. 47:267–275.
23. Gulati, B. R., R. Deepa, B. K. Singh, and C. Durga Rao. 2007. Diversity in
Indian equine rotaviruses: identification of genotype G10, P6 and G1
strains and a new VP7 genotype (G16) strain in specimens from diarrheic
foals in India. J. Clin. Microbiol. 45:972–978.
24. Gunasena, S., O. Nakagomi, Y. Isegawa, E. Kaga, T. Nakagomi, A. D. Steele,
J. Flores, and S. Ueda. 1993. Relative frequency of VP4 gene alleles among
human rotaviruses recovered over a 10-year period (1982–1991) from Jap-
anese children with diarrhea. J. Clin. Microbiol. 31:2195–2197.
25. Hoshino, Y., M. M. Sereno, K. Midthun, J. Flores, A. Z. Kapikian, and R. M.
Chanock. 1985. Independent segregation of two antigenic specificities (VP3
and VP7) involved in neutralization of rotavirus infectivity. Proc. Natl. Acad.
Sci. USA 82:8701–8704.
26. Hoshino, Y., and A. Z. Kapikian. 2000. Rotavirus serotypes: classification
and importance in epidemiology, immunity, and vaccine development.
J. Health Popul. Nutr. 18:5–14.
27. Iturriza-Gomara, M., J. Green, D. W. Brown, U. Desselberger, and J. J.
Gray. 2000. Diversity within the VP4 gene of rotavirus P strains: implica-
tions for reverse transcription-PCR genotyping. J. Clin. Microbiol. 38:898–
28. Iturriza-Gomara, M., G. Kang, and J. Gray. 2004. Rotavirus genotyping:
keeping up with an evolving population of human rotaviruses. J. Clin. Virol.
29. Jiang, B., J. R. Gentsch, and R. I. Glass. 2002. The role of serum antibodies
in the protection against rotavirus disease: an overview. Clin. Infect. Dis.
30. Kapikian, A. Z., Y. Hoshino, and R. M. Chanock. 2001. Rotavirues, p.
1787–1833. In D. M. Knipe, P. M. Howley, D. E. Griffin, R. A. Lamb, M. A.
Martin, B. Roizman, and S. E. Straus (ed.), Fields virology, 4th ed. Lippin-
cott Williams & Wilkins, Philadelphia, PA.
31. Kawaguchi, K., S. Kaneko, M. Honda, H. F. Kawai, Y. Shirota, and K.
Kobayashi. 2003. Detection of hepatitis B virus DNA in sera from patients
with chronic hepatitis B virus infection by DNA microarray method. J. Clin.
32. Koshimura, Y., T. Nakagomi, and O. Nakagomi. 2000. The relative frequen-
cies of G serotypes of rotaviruses recovered from hospitalized children with
diarrhea: a 10-year survey (1987–1996) in Japan with a review of globally
collected data. Microbiol. Immunol. 44:499–510.
33. Li, J., S. Chen, and D. H. Evans. 2001. Typing and subtyping influenza virus
VOL. 45, 2007GENOTYPING OF ROTAVIRUSES BY MICROARRAY ANALYSIS 2647
using DNA microarrays and multiplex reverse transcriptase PCR. J. Clin. Download full-text
34. Lin, B., G. J. Vora, D. Thach, E. Walter, D. Metzgar, C. Tibbetts, and D. A.
Stenger. 2004. Use of oligonucleotide microarrays for rapid detection and
serotyping of acute respiratory disease-associated adenoviruses. J. Clin. Mi-
35. Lovmar, L., C. Fock, F. Espinoza, F. Bucardo, A. C. Syvanen, and K. Bondeson.
2003. Microarrays for genotyping human group A rotavirus by multiplex
capture and type-specific primer extension. J. Clin. Microbiol. 41:5153–5158.
36. Martella, V., M. Ciarlet, K. Banyai, E. Lorusso, S. Arista, A. Lavazza, G.
Pexxotti, N. Decaro, A. Cavalli, M. S. Lucente, M. Corrente, G. Elia, M.
Camero, M. Tempesta, and C. Buonavoglia. 2007. Identification of group A
porcine rotavirus strains bearing a novel VP4 (P) genotype in Italian swine
herds. J. Clin. Microbiol. 45:577–580.
37. Martella, V., M. Ciarlet, K. Banyai, E. Lorusso, A. Cavalli, M. Corrente, G.
Elia, S. Arista, M. Camero, C. Desario, N. Decaro, A. Lavazza, and C.
Buonavoglia. 2006. Identification of a novel VP4 genotype carried by a
serotype G5 porcine rotavirus strain. Virology 46:301–311.
38. Martella, V., V. Terio, S. Arista, G. Elia, M. Corrente, A. Madio, A. Pratelli,
M. Tempesta, G. Cirani, and C. Buonavoglia. 2004. Nucleotide variation in
the VP7 gene affects PCR genotyping of G9 rotaviruses identified in Italy.
J. Med. Virol. 72:143–148.
39. Neverov, A. A., M. A. Riddell, W. J. Moss, D. V. Volokhov, P. A. Rota, L. W.
Lowe, D. Chibo, S. B. Smit, D. E. Griffin, K. M. Chumakov, and V. E.
Chizhikov. 2006. Genotyping of measles virus in clinical specimens on the
basis of oligonucleotide microarray hybridization patterns. J. Clin. Microbiol.
40. Nordstrom, H., P. Johansson, Q. G. Li, A. Lundkvist, P. Nilsson, and F.
Elgh. 2004. Microarray technology for identification and distinction of han-
taviruses. J. Med. Virol. 72:646–655.
41. Nordstrom, H., K. I. Falk, G. Lindegren, J. Mouzavi-Jazi, A. Walden, F.
Elgh, P. Nilsson, and A. Lundkvist. 2005. DNA microarray technique for
detection and identification of seven flaviviruses pathogenic for man. J. Med.
42. Offit, P. A., and G. Blavat. 1986. Identification of the two rotavirus genes
determining neutralization specificities. J. Virol. 57:376–378.
43. Parashar, U. D., C. J. Gibson, J. S. Bresee, and R. I. Glass. 2006. Rotavirus
and severe childhood diarrhea. Emerg. Infect. Dis. 12:304–306.
44. Parra, G. I., and E. E. Espinola. 2006. Nucleotide mismatches between the
VP7 gene and the primer are associated with genotyping failure of a specific
lineage from G1 rotavirus strains. Virol. J. 3:35.
45. Petricoin, E. F., III, J. L. Hackett, L. J. Lesko, P. K. Puri, S. I. Gutman, K.
Chumakov, J. Woodcock, D. W. Feigal, Jr., K. C. Zoon, and F. D. Sistare.
2002. Medical applications of microarray technologies: a regulatory science
perspective. Nat. Genet. 32(Suppl.):474–479.
46. Rahman, M., J. Matthijnssens, S. Nahar, G. Podder, D. A. Sack, T. Azim,
and M. Van Ranst. 2005. Characterization of a novel P, G11 group A
rotavirus. J. Clin. Microbiol. 43:3208–3212.
47. Rahman, M., J. Matthijnssens, X. Yang, T. Delbeke, I. Arijs, K. Taniguchi,
M. Iturriza-Gomara, N. Iftekharuddin, T. Azim, and M. Van Ranst. 2007.
Evolutionary history and global spread of the emerging G12 human rotavi-
ruses. J. Virol. 81:2382–2390.
48. Rahman, M., R. Sultana, G. Podder, A. S. Faruque, J. Matthijnssens, K.
Zaman, R. F. Breiman, D. A. Sack, M. Van Ranst, and T. Azim. 2005. Typing
of human rotaviruses: nucleotide mismatches between the VP7 gene and
primer are associated with genotyping failure. Virol. J. 2:24.
49. Ramsay, G. 1998. DNA chips: state-of-the art. Nat. Biotechnol. 16:40–44.
50. Ryabinin, V. A., L. A. Shundrin, E. B. Kostina, M. Laassri, V. Chizhikov,
S. N. Shcheljunov, K. Chumakov, and A. N. Sinyakov. 2006. Microarray
assay for detection and discrimination of Orthopoxvirus species. J. Med.
51. Santos, N., and Y. Hoshino. 2005. Global distribution of rotavirus serotypes/
genotypes and its implication for the development and implementation of an
effective rotavirus vaccine. Rev. Med. Virol. 25:29–56.
52. Santos, N., E. M. Volotao, C. C. Soares, M. C. Albuquerque, F. M. da Silva,
V. Chizhikov, and Y. Hoshino. 2003. VP7 gene polymorphism of serotype G9
rotavirus strains and its impact on G genotype determination by PCR. Virus
53. Santos, N., E. M. Volotao, C. C. Soares, M. C. Albuquerque, F. M. da Silva,
T. R. de Carvalho, C. F. Pereira, V. Chizhikov, and Y. Hoshino. 2001.
Rotavirus strains bearing genotype G9 and P recovered from Brazilian
children with diarrhea from 1997 to 1999. J. Clin. Microbiol. 39:1157–1160.
54. Sergeev, N., E. Rubtcova, V. Chizhikov, D. S. Schmid, and V. N. Loparev.
2006. New mosaic subgenotype of varicella-zoster virus in the USA: VZV
detection and genotyping by oligonucleotide-microarray. J. Virol. Methods
55. Shaw, R. D., D. L. Stoner-Ma, M. K. Estes, and H. B. Greenberg. 1985.
Specific enzyme-linked immunoassay for rotavirus serotypes 1 and 3. J. Clin.
56. Steyer, A., M. Poljsak-Prijatelj, D. Barlic-Maganja, U. Jamnikar, J. Z.
Mijovski, and J. Marin. 2007. Molecular characterization of a new porcine
rotavirus P genotype found in an asymptomatic pig in Slovenia. Virology
57. Taniguchi, K., T. Urasawa, Y. Morita, H. B. Greenberg, and S. Urasawa.
1987. Direct serotyping of human rotavirus in stools by an enzyme-linked
immunosorbent assay using serotype 1-, 2-, 3-, and 4-specific monoclonal
antibodies to VP7. J. Infect. Dis. 155:1159–1166.
58. Taniguchi, K., F. Wakasugi, Y. Pongsuwanna, T. Urasawa, S. Ukae, S.
Chiba, and S. Urasawa. 1992. Identification of human and bovine rotavirus
serotypes by polymerase chain reaction. Epidemiol. Infect. 109:303–312.
59. Wang, D., L. Coscoy, M. Zylberberg, P. C. Avila, H. A. Boushey, D. Ganem,
and J. L. DeRisi. 2002. Microarray-based detection and genotyping of viral
pathogens. Proc. Natl. Acad. Sci. USA 99:15687–15692.
60. Wyatt, R. G., H. B. Greenberg, W. D. James, A. L. Pittman, A. R. Kalica, J.
Flores, R. M. Chanock, and A. Z. Kapikian. 1982. Definition of human
rotavirus serotypes by plaque reduction assay. Infect. Immun. 37:110–115.
2648HONMA ET AL. J. CLIN. MICROBIOL.