Virology 325 (2004) 11 – 17

www.elsevier.com/locate/yviro

Human infection with a P[14], G3 lapine rotavirus

Karolien De Leener, a Mustafizur Rahman, a,b Jelle Matthijnssens, a Lieve Van Hoovels, a
Truus Goegebuer, a Ingrid van der Donck, a and Marc Van Ranst a,*
a
Laboratory of Clinical and Epidemiological Virology, Rega Institute for Medical Research, University of Leuven, BE-3000 Louvain, Belgium
b
ICDDR,B: Centre for Health and Population Research, Mohakhali, Dhaka 1212, Bangladesh

Received 21 November 2003; returned to author for revision 7 January 2004; accepted 12 April 2004

Abstract

Group A rotaviruses are the main cause of severe diarrhoea in humans and animals throughout the world. We report the first description of
a clinically apparent infection with a P[14], G3 rotavirus (strain B4106) in a hospitalized 6-year-old child. The VP7 gene of the B4106 strain
had the closest sequence similarity (94% and 97% on the nucleotide and amino acid level, respectively) with strain 30/96 (P[14], G3), a
lapine rotavirus isolated in an Italian rabbit in 1996 while the VP4 gene had the closest similarity with strain 30/96 on the nucleotide level
(96%), and with lapine strains C-11 (P[14], G3) and Alabama (P[14], G3), isolated in the United States in the 1980s on the amino acid level
(99%). The host restriction determinant gene NSP4 of B4106 was also most similar to lapine strain Alabama (95% nt identity and 97% aa
identity). Phylogenetic analysis showed that the VP4, VP7, and NSP4 genes of the B4106 strain share a common evolutionary lineage with
those of lapine rotavirus strains. We therefore hypothesize that a lapine rotavirus was able to cross the host species barrier and caused disease
in a new host. The increasing detection of strains in humans that were previously believed to be restricted to animals raises questions whether
interspecies transmission of rotaviruses is a common event in nature.
D 2004 Elsevier Inc. All rights reserved.

Keywords: Rotavirus; Diarrhea; Rabbit; Interspecies transmission; VP7; VP4; NSP4; Phylogeny; Sequencing; G type; P type

Introduction and P combinations (Kapikian et al., 1996; Liprandi et al.,

Group A rotaviruses are the most common cause of acute
infectious diarrhea in humans and in a wide variety of
mammalian and avian species. Rotavirus-associated diar-
rhea annually leads to more than 125 million cases of
infantile gastroenteritis and 352,000 – 592,000 deaths in
children under 5 years old, primarily in less-developed
countries (Parashar et al., 2003). Rotaviruses belong to the
Reoviridae and have a segmented double-stranded RNA
genome (11 segments). A dual classification system has
been established for group A rotaviruses, with the viral
capsid glycoprotein VP7 defining G types and the protease
sensitive protein VP4 defining P types. At least 15 G types
and 23 P types have been isolated globally with various G
* Corresponding author. Laboratory of Clinical and Epidemiological
Virology, Department of Microbiology and Immunology, Rega Institute for
Medical Research, Minderbroedersstraat 10, BE-3000 Louvain, Belgium.
Fax: +32-16-332131.
E-mail address: marc.vanranst@uz.kuleuven.ac.be (M. Van Ranst).
2003; Martella et al., 2003; Okada et al., 2000; Rao et al.,
2000; Sereno and Gorziglia, 1994). Because VP7 and VP4
are encoded by different RNA segments, various combina-
tions of G and P types can be observed. The four major
human G types are G1, G2, G3, and G4, and the less
common types are G5, G8, G9, and G12 (Bishop, 1996;
Green et al., 1989; Griffin et al., 2002; Kang et al., 2002;
Santos et al., 2001). G6 and G10 rotaviruses are the two
major types isolated from cattle, but have been infrequently
encountered in humans (Banyai et al., 2003). Until now, G7
(chicken and cattle), G11 (pigs), G13 and G14 (horses), and
G15 (cattle) rotavirus strains have not been isolated from
humans (Browning et al., 1991a, 1991b; Rao et al., 2000).
In humans, P[8] is the most common genotype detected
worldwide, followed by P[4] and P[6] (Estes, 1996; Gentsch
et al., 1996).
Rotaviruses are the only known agents having 11 seg-
ments of dsRNA that infect humans (Kapikian et al.,
1996). Genetic reassortment is facilitated by the segmented
nature of the rotavirus genome, and significantly contrib-

0042-6822/$ - see front matter D 2004 Elsevier Inc. All rights reserved.
doi:10.1016/j.virol.2004.04.020
12 K. De Leener et al. / Virology 325 (2004) 11–17

has also been detected in goats (de Beer and Steele,

unpublished data, accession number AY128708).
Bryden et al. (1976) first isolated rotaviruses from rabbits
in 1976. Sato et al. (1982) isolated the R-2 and R-3 strains
from an outbreak of lapine diarrhea during 1975– 1978 in
Japan, and identified them as serologically distinct from other
rotaviruses. Thouless et al. (1986) confirmed the isolates as
G3 types in 1985, but the P type remained undetermined until
Ciarlet et al. confirmed the R-2 strain as P[14] in 1997. Most
of the lapine rotaviruses isolated so far including the latest
reported case (strain 30/96 isolated in 1996) contain similar
VP7 and VP4 gene combination (P[14], G3) (Ciarlet et al.,
1997; Martella et al., 2003). There may exist two lapine G3
subtypes, one represented by the American lapine strains
isolated in 1983– 1989 and the other by the Japanese R-2
strain isolated in 1975– 1978 (Ciarlet et al., 1997).
We describe a Belgian rotavirus strain (B4106) that
caused severe diarrhea in a hospitalized child. This is the
Fig. 1. Electropherotype comparison between our B4106 strain and human first reported case of a lapine rotavirus in a human. We
G1 and G9 rotavirus strains by polyacrylamide gel electrophoresis and investigated the evolutionary relationship of this strain
silver staining of the 11 rotaviral RNA segments. with other G3 and P[14] rotaviruses by comparing the
nucleotide sequences and by phylogenetic analysis of VP4,
utes to the genetic diversity of rotaviruses. Reassortment VP7, and NSP4 genes available in GenBank.
during co-infection with different rotaviruses can lead to
progeny viruses with novel or atypical phenotypes that
inherit genome segments from both parental strains (Pal- Table 1
ombo, 2002). This unique mechanism leads to interspecies Nucleotide and amino acid sequence similarity of strain B4106 with VP7
transmission events between human and animal rotavi- sequences with different G genotype specificities
ruses. Interspecies transmission through reassortment was G type Strain Origin Similarity (%)
documented more frequently than through transfer of the Nucleotide Amino acid
whole virus gene constellation. The increasing number of
G1 Wa human 72 85
reports of novel rotavirus strains in humans suggests that G2 HU5 human 72 73
interspecies transmissions of whole virus strain or individ- G3 SA11 simian 81 93
ual gene segment(s) greatly contribute to the overall G3 A131 porcine 79 90
rotavirus diversity (reviewed by Palombo, 2002). G3 K9 canine 87 94
Rotavirus G3 strains were considered to be intraserotypi- G3 ERV316 equine 86 94
G3 Ch-32 human 74 82
cally more heterologous than G1, G2, and G4 rotaviruses G3 J-12 human 81 90
(Wen et al., 1997). Thus far, G3 types were isolated from G3 T108 human 78 90
humans, rabbits, pigs, birds, cats, dogs, monkeys, horses, G3 RRV rhesus 83 96
mice, cows, and lambs (Gerna et al., 1992; Hoshino et al., G3 160/01 lapine 94 96
1984). It is the only rotavirus G type for which such a broad G3 30/96 lapine 94 97
G3 BAP-2 lapine 90 97
host range has been described. A close evolutionary rela- G3 229/01 lapine 94 97
tionship between G3 and G14 rotaviruses has been predicted G3 J63 bovine 83 95
by RNA – RNA hybridization (Nakagomi et al., 2003). G4 ST3 human 75 78
There are as few as six differing amino acid residues in G5 OSU porcine 76 84
the VP7 antigenic regions of strain FI23 (a prototype G14 G6 NCDV bovine 74 83
G7 Ch2 chicken 73 61
equine rotavirus) and G3 rotaviruses (Browning et al., G8 B37 human 76 80
1991b). G9 116E human 76 84
Rotavirus P[14] was described in rabbits, humans, and G10 B223 bovine 75 81
goats. The first human P[14] strain (G6) was reported by G11 YM porcine 77 88
Gerna et al. in Italy in 1992. Human P[14] rotaviruses have G12 L26 human 75 80
G13 L338 equine 76 82
also been isolated in Finland (G8), Thailand (G10), South G14 FR4 equine 79 85
Africa (G1), Australia (G6), Egypt (G8), and Hungary (G6) G14 CH3 equine 80 84
(Banyai et al., 2003; Gerna et al., 1994; Holmes et al., 1999; G14 FR8 equine 78 85
Mphahlele and Steele, 1995; Palombo and Bishop, 1995; G14 FI23 equine 79 87
Urasawa et al., 1993). A single P[14] strain (Cap455, G6) G15 Hg18 bovine 75 77
 K. De Leener et al. / Virology 325 (2004) 11–17 13

Results and discussions
Case history and rotavirus antigen detection
A 6-year-old Belgian child was admitted in 2000 to the
Gasthuisberg University Hospital in Leuven, Belgium, with
nausea and severe diarrhea. After several days of passing
watery stools, progressive dehydration necessitated intrave-
nous rehydration therapy. The further recovery of the child
was uneventful, and he could leave the hospital after 2 days.
A stool specimen (B4106) was found positive for rotavirus
using a commercial rotavirus antigen solid-phase sandwich-
type enzyme immunoassay (Premier Rotaclone, Meridian
Bioscience, Cincinnati, OH). Further characterization of the
sample through RT-PCR and sequencing of the partial VP7
and VP4 gene sequence revealed a P[14], G3 type-specific
rotavirus.
RNA pattern (electropherotype)
Viral RNA was extracted from the stool sample and was
run in polyacrylamide gel electrophoresis. A unique RNA
migration pattern was observed for B4106, which was also
observed in the Alabama strain isolated from a rabbit in the
United States. The gene segment 11 of the Alabama strain had
a ‘‘super short’’ RNA pattern (Gorziglia et al., 1989). The
RNA segment 11 is longer than those reported for rotavirus
strains with a long electropherotype. Fig. 1 shows the electro-
pherotype comparison between our B4106 strain and G1 and
G9 human rotaviruses isolated in Belgium.
VP7 sequence analysis and determination of G genotype
The partial nucleotide (1007 bp) and deduced amino acid
sequence of the VP7-encoding gene of the B4106 strain was
determined (GenBank accession number AY456382) and
compared with VP7 sequences of prototype strains belonging
to G1 to G15 (Table 1). Sequence comparison indicated that
the VP7 sequence of strain B4106 was most closely related to
the VP7 of the lapine G3 rotavirus strains (90 –94% nucle-
otide and 96 –97% amino acid identity), with the highest
similarity with the Italian lapine G3-rotavirus strain 30/96
(94% nt and 97% aa identity). Rotavirus strains representing
other G types exhibited much less nucleotide and amino acid
identity (72 – 80% nt and 61– 87% aa identity) with our strain.
We found that the lapine G3 strains showed almost the same
nt and aa identity with equine G14 rotaviruses (78 – 80% nt
and 84– 87% aa identity) as with the human G3 rotaviruses

Fig. 2. Neighbor-joining phylogenetic tree based on nucleotide sequences of the VP7 encoding genes for B4106 and other established rotavirus G3 genotypes.
The VP7 sequences were obtained from published reports and the GenBank database. La, lapine; Hu, human; Ca, caprine; Po, porcine; Bo, bovine; Eq, equine;
Rh, rhesus. The different strains were named as per their GenBank accession numbers. The numbers adjacent to the nodes represent the percentage of bootstrap
support (of 1000 replicates) for the clusters to the right of the node. Bootstrap values lower than 70% were not shown. The dendrogram is rooted using human
G1, G4, and G6 rotaviruses. Equine G14 rotaviruses, which are genetically closely related to G3 rotaviruses, were also included.
14 K. De Leener et al. / Virology 325 (2004) 11–17

(74 –81% nt and 82– 90% aa identity). A phylogenetic tree Table 2

Nucleotide and amino acid sequence similarity of strain B4106 with VP4
was constructed that included all known VP7 sequences of
sequences with different P genotype specificities
G3 and related G14 rotaviruses in the GenBank database
P type Strain Origin Similarity (%)
(release 130.0) (Fig. 2). Two major lineages were found
among G3 rotaviruses. One lineage clusters the G3 human Nucleotide Amino acid
rotaviruses with some bovine and porcine rotaviruses. The P[1] A5 bovine 63 60
other lineage consists of all nonhuman G3 rotaviruses with P[2] SA11 simian 63 60
P[3] HCR3 human 60 58
the exception of two human rotaviruses strain B4106 and
P[4] RV5 human 56 55
strain HCR3 (accession number L21666). The phylogenetic P[5] UK bovine 57 57
analysis confirmed that our B4106 strain clustered with the P[6] Gottfried porcine 73 54
lapine rotavirus strains in a separate branch with other animal P[7] OSU porcine 57 60
(canine, equine, bovine, rhesus) G3 rotaviruses. This branch P[8] Wa human 60 54
P[9] AU1 human 74 83
is only remotely related to the other human G3 rotaviruses. It
P[10] 69M human 59 61
was also interesting to see that equine G14 rotaviruses share a P[11] KK-3 bovine 71 43
common ancestor with the animal G3 rotaviruses. P[12] H2 equine 62 59
P[13] MDR13 porcine 75 57
VP4 sequence analysis and determination of P genotype P[14] Mc35 human 80 91
P[14] PA169 human 81 93
P[14] C-11 lapine 96 99
We could not amplify the VP4 gene of our B4106 strain P[14] Alabama lapine 95 99
with general VP4 primers (Con3-Con2), and a new primer set P[14] BAP-2 lapine 94 98
Con3L and Con2L was developed for the amplification of an P[14] 30/96 lapine 96 96
877-bp fragment of the VP4 gene. The partial VP4-encoding P[14] R2 lapine 81 89
P[15] LP14 ovine 63 58
gene sequence (838 bp; accession number AY456383) of
P[16] Eb murine 69 58
strain B4106 was compared with 23 other P genotypes (Table P[17] 993/83 bovine 76 47
2). Strain B4106 showed the highest similarity to P[14] P[18] L338 equine 65 62
rotavirus types, with the greatest identity to lapine rotavirus P[19] 4F porcine 69 54
strain 30/96 (96% nt identity). On the deduced amino acid P[20] EHP murine 73 57
P[21] Hg18 bovine 60 60
level, the most similar VP4 sequence in comparison to our
P[22] 160/01 lapine 66 57
B4106 strain was C-11 (99% aa identity), a lapine rotavirus P[23] A34 porcine 60 56
isolated in the United States in 1983. Non-P[14] rotavirus
strains showed only 57 – 76% nt identity and 43 –83% aa
identity with the B4106 strain in the VP4 region. A dendro- distantly related to human and other animal rotavirus strains
gram was constructed based on the VP4 partial deduced (data not shown).
amino acid sequences of B4106 and other P[14] rotaviruses
found in GenBank (Fig. 3). The phylogenetic tree clustered Origin of the B4106 strain
the P[14] rotavirus strains in three different clusters. Cluster I
(animal cluster) originated from a Japanese lapine strain R-2 Amino acid sequence comparisons, phylogenetic anal-
that was isolated in the 1970s. Our strain B4106 (P[14], G3) yses of VP4, VP7 and NSP4 genes, and electropherotyp-
belongs to cluster I with five lapine P[14], G3 strains. Cluster ing indicated that strain B4106 shares most of the genetic
II contains human P[14] strains (and one caprine strain) that characteristics with lapine rotavirus strains. All rotaviruses
are associated with either the G6 or G8 VP7 gene segment. A that were isolated from rabbits contain the unique VP4/
more distantly related P[14], G10 strain (Cluster III: Mc35) VP7 gene combination P[14], G3. No other strain from
was isolated in Thailand in the 1990s (Urasawa et al., 1993). human or animal origin contained this P[14], G3 gene
combination. The B4106 strain is most similar to the
NSP4 sequence analysis lapine rotavirus strain 30/96 isolated in Italy in 1996, and
they clustered together in the same branches of the G3
A new primer set (LAP_NSP4_1F and LAP_NSP4_751- and P[14] phylogenetic trees. It is possible that the P[14],
R) was developed based on the alignment of several lapine G3 strain is circulating in the rabbit population in
NSP4 sequences found in GenBank. The 10th gene segment, European countries and that it crossed the host species
encoding nonstructural protein NSP4 of B4106 was ampli- barriers infecting a human child. This hypothesis is
fied and sequenced (accession number AY576006) and the supported by the finding of other interspecies transmis-
deduced amino acid sequence was analyzed. The greatest sion events of G3 rotaviruses from canine and feline
similarity of the NSP4 gene of B4106 was with the lapine origin to humans (Li et al., 1994; Nakagomi and Naka-
strain Alabama (95% nt identity and 97% aa identity). gomi, 2000; Nakagomi et al., 1992).
Phylogenetic analysis also showed that the NSP4 gene of The NSP4 of B4106 clustered together with other lapine
B4106 clustered with lapine rotavirus strains and was only rotavirus strains and was distantly related to human and other
 K. De Leener et al. / Virology 325 (2004) 11–17 15

Fig. 3. Neighbor-joining phylogenetic tree based on amino acid sequences of the VP4 encoding genes for B4106 and other established rotavirus P[14]
genotypes. The VP4 gene sequences were obtained from published reports and the GenBank database. BEL, Belgium; JAP, Japan; EGY, Egypt; ITA, Italy; SA,
South Africa; AUS, Australia; HUN, Hungary. The GenBank accession numbers were: C-11 (U62150), BAP-2 (U62151), Alabama (U62149), R-2 (U62152),
HAL1166 (L20875), PA169 (D14724), MG6 (U22012), Cap455 (AY128709), EGY2295 (AF104103), Mc35 (D14032), AU-1 (D10970), Hun5 (AJ488139),
30/96 (AF526376). The numbers adjacent to the nodes represent the percentage of bootstrap support (of 1000 replicates) for the clusters to the right of the node.
Bootstrap values lower than 70% were not shown. The dendrogram is rooted using a human rotavirus P[9]G3.

animal rotavirus strains. It was suggested by Iturriza-Gomara
et al. (2003) that this protein might be a host restriction
determinant. Ciarlet et al. (2000) also described that within
NSP4 genotypes, strains isolated from different hosts clus-
tered according to species of origin, suggesting a conserved
pattern of evolution within species. It is likely that the finding
of the B4106 strain in the infected child was an interspecies
transmission event through whole virus transmission from
rabbit to human rather than through reassortment of gene
segments from different rotaviruses. Another observation in
support of this hypothesis was the finding that the child lived
in a rural environment close to grasslands where rabbits were
very frequently encountered. Surveillance of rotavirus strains
may need to be expanded to include domestic animal and
avian species as they may serve as a reservoir for viral
reassortment, interspecies transmission, and evolution of
novel rotavirus strains.
enzyme immunoassay (Meridian Bioscience, Cincinnati,
OH). An aliquot of a fecal suspension was added to a
plastic microtiter well coated with a monoclonal antibody
directed against the rotavirus group-specific antigen
(VP6). The solution was simultaneously incubated with
an anti-rotavirus monoclonal antibody conjugated to
horseradish peroxidase, resulting in the rotavirus antigen
being sandwiched between the solid-phase and the en-
zyme-linked antibodies. After 60-min incubation at room
temperature, the sample well was washed to remove
unbound enzyme-labeled antibodies. Urea peroxide and
tetramethylbenzidine were added as substrate and incu-
bated for 10 min at room temperature. The enzymatic
reaction that converts the colorless substrate to a blue
color was stopped with 1 N H2SO4, and the absorbance
was determined spectrophotometrically. Specimens with
absorbance units (A450) greater than 0.150 were consid-
ered positive.

Materials and methods Polyacrylamide gel electrophoresis

Rotavirus antigen detection The electropherotype (e-type) of the 11 gene segments

of B4106 was investigated using polyacrylamide gel
Rotavirus antigen was detected in the stool specimen electrophoresis (PAGE) as described by Herring et al.
using the Premier Rotaclone solid-phase sandwich-type (1982).
16 K. De Leener et al. / Virology 325 (2004) 11–17
RNA extraction
Viral RNA was extracted from 140 Al of the feces sample
using the QIAamp Viral RNA mini kit (Qiagen/Westburg,
Leusden, The Netherlands) according to the manufacturer’s
instructions.
RT-PCR
The extracted RNA was denatured at 97 jC for 5 min.
Reverse transcriptase PCR (RT-PCR) was carried out
using the Qiagen OneStep RT-PCR Kit (Qiagen/West-
burg). For G typing, we amplified a 1062-bp fragment of
the VP7 gene with the forward primer Beg9 (5V-
GGCTTTAAAAGAGAGAATTTCCGTCTGG-3V; proto-
type strain Wa, GenBank accession number M21843, nt
1 –28) and the reverse primer End9 (5V-GGTCACATCA-
TACA ATTCTAATCTAAG-3V; prototype strain SA11,
accession number K02028, nt 1062– 1036). For P typing,
an 877-bp fragment of the VP4 gene was amplified using
forward primer Con3L (5V-TGGCTTCTTTGATTTA-
TAGGCA-3V; prototype strain BAP-2, accession number
U62151, nt 11 – 32) and reversed primer Con2L (5V-
ATTTCTGACAATTTATATCC-3V; prototype strain BAP-
2, nt 887– 868). For the amplification of the 751 bp of
the NSP4 gene, forward primer LAP_NSP4_1F (5V-
GGCTTTTAAAAGTTCTGTTCC-3V prototype strain
ALA, accession number AF144792, nt 1– 21) and reverse
primer LAP _ NSP4 _ 751R (5V-GGTCACACTAAGAC-
CATTCC-3V, prototype strain ALA, nt 751– 732) were
used.
The RT-PCR reaction was carried out with an initial
reverse transcription step at 45 jC for 30 min, followed
by PCR activation at 95 jC for 15 min, 35 cycles of
amplification (30 s at 94 jC, 45 s at 45 jC, 1 min at
72 jC), and a final extension of 7 min at 72 jC in a
GeneAmp PCR System 9700 thermal cycler (Perkin-
Elmer Applied Biosystems, Foster City, CA, USA).
PCR products were run on a polyacrylamide gel,
stained with ethidium bromide, and visualized under
UV light.
Nucleotide sequencing
The PCR amplicons were purified with the QIA
quick PCR purification kit (Qiagen/Westburg) and se-
quenced in both directions using the dideoxy-nucleotide
chain termination method with the ABI PRISM BigDye
Terminator Cycle Sequencing Reaction kit (Perkin-Elmer
Applied Biosystems) on an automated sequencer (ABI
PRISM 3100) at the Rega Institute core sequencing
facility. The Beg9 and End9 RT-PCR primers (for the
VP7 gene), the Con2L and Con3L primers (for the VP4
gene), and the LAP_NSP4_1F and LAP_NSP4_751R
primers (for the NSP4 gene) were also used as se-
quencing primers.
DNA and protein sequence analysis
The chromatogram sequencing files were inspected using
Chromas 2.23 (Technelysium, Queensland, Australia), and
contigs were prepared using SeqMan II (DNASTAR, Mad-
ison, WI). Nucleotide and protein sequence similarity
searches were performed using the National Center for
Biotechnology Information (NCBI, National Institutes of
Health, Bethesda, MD) BLAST (Basic Local Alignment
Search Tool) server on GenBank database release 130.0
(Altschul et al., 1990). Pairwise sequence alignments were
performed using the Sequence Analysis Server at Michigan
Technological University (http://genome.cs.mtu.edu), and
multiple sequence alignments were calculated using CLUS-
TALX 1.81 (Thompson et al., 1997). Sequences were
manually edited in the GeneDoc version 2.6.002 alignment
editor (Nicholas et al., 1997).
Phylogenetic analysis
Phylogenetic and molecular evolutionary analyses were
conducted using the MEGA version 2.1 software package
(Kumar et al., 2001), based on the different G3, G14, P[14],
and lapine NSP4 rotavirus sequences available in GenBank
version 130.0. Genetic distances were calculated using the
Kimura-2 parameter. The dendrograms were constructed
using the neighbor-joining method.
DNA sequence submission
The nucleotide sequence data reported in this paper
were deposited in GenBank using the National Center for
Biotechnology Information (NCBI) BankIt v3.0 submis-
sion tool (http://www3.ncbi.nlm.nih.gov/BankIt/) under ac-
cession numbers AY456382 (for the VP7 gene),
AY456383 (for the VP4 gene) and AY576006 (for the
NSP4 gene).
References
Altschul, S., Gish, F.W., Miller, W., Myers, E.W., Lipman, D.J., 1990.
Basic local alignment search tool. J. Mol. Biol. 215, 403 – 410.
Banyai, K., Gentsch, J.R., Griffin, D.D., Holmes, J.L., Glass, R.I., Szucs,
G., 2003. Genetic variability among serotype G6 human rotaviruses:
identification of a novel lineage isolated in Hungary. J. Med. Virol. 71,
124 – 134.
Bishop, R.F., 1996. Natural history of human rotavirus infection. Arch.
Virol. 12, 119 – 128.
Browning, G.F., Chalmers, R.M., Fitzgerald, T.A., Snodgrass, D.R., 1991a.
Serological and genomic characterization of L338, a novel equine group
A rotavirus G serotype. J. Gen. Virol. 72, 1059 – 1064.
Browning, G.F., Fitzgerald, T.A., Chalmers, R.M., Snodgrass, D.R., 1991b.
A novel group A rotavirus G serotype: serological and genomic char-
acterization of equine isolate FI23. J. Clin. Microbiol. 29, 2043 – 2046.
Bryden, A.S., Thouless, M.E., Flewett, T.H., 1976. Rotavirus and rabbits.
Vet. Rec. 99, 323.
Ciarlet, M., Estes, M.K., Conner, M.E., 1997. Comparative amino acid
sequence analysis of the outer capsid protein VP4 from four lapine
 K. De Leener et al. / Virology 325 (2004) 11–17
rotavirus strains reveals identity with genotype P[14] human rotavi-
ruses. Arch. Virol. 142, 1059 – 1069.
Ciarlet, M., Liprandi, F., Conner, M.E., Estes, M.K., 2000. Species spec-
ificity and interspecies relatedness of NSP4 genetic groups by compar-
ative NSP4 and sequence analysis of animal rotaviruses. Arch. Virol.
145, 371 – 383.
Estes, M.K., 1996. Rotaviruses and their replication. In: Fields, B.N.,
Knipe, D.M., Howley, P.M. (Eds.), Fields Virology, 3rd ed, vol. 2.
Lippincott-Raven Publishers, Philadelphia, pp. 1625 – 1655.
Gentsch, J.R., Woods, P.A., Ramachandran, M., Das, B.K., Leite, J.P.,
Alfieri, A., Kumar, R., Bhan, M.K., Glass, R.I., 1996. Review of G
and P typing results from a global collection of rotavirus strains: impli-
cations for vaccine development. J. Infect. Dis. 174 (Suppl. 1), S30 – S36.
Gerna, G., Sarasini, A., Parea, M., Arista, S., Miranda, P., Brussow, H.,
Hoshino, Y., Flores, J., 1992. Isolation and characterization of two
distinct human rotavirus strains with G6 specificity. J. Clin. Microbiol.
30, 9 – 16.
Gerna, G., Steele, A.D., Hoshino, Y., Sereno, M., Garcia, D., Sarasini, A.,
Flores, J., 1994. A comparison of the VP7 gene sequences of human
and bovine rotaviruses. J. Gen. Virol. 75, 1781 – 1784.
Gorziglia, M., Nishikawa, K., Fukuhara, N., 1989. Evidence of duplication
and deletion in super short segment 11 of rabbit rotavirus Alabama
strain. Virology 170, 587 – 590.
Green, K.Y., Hoshino, Y., Ikegami, N., 1989. Sequence analysis of the gene
encoding the serotype-specific glycoprotein (VP7) of two new human
rotavirus serotypes. Virology 168, 429 – 433.
Griffin, D.D., Nakagomi, T., Hoshino, Y., Nakagomi, O., Kirkwood, C.D.,
Parashar, U.D., Glass, R.I., Gentsch, J.R., 2002. Characterization of
nontypeable rotavirus strains from the United States: identification of
a new rotavirus reassortant (P2A[6],G12) and rare P3[9] strains related
to bovine rotaviruses. Virology 294, 256 – 269.
Herring, A.J., Inglis, N.F., Ojeh, C.K., Snodgrass, D.R., Menzies, J.D.,
1982. Rapid diagnosis of rotavirus infection by direct detection of viral
nucleic acid in silver-stained polyacrylamide gels. J. Clin. Microbiol.
16, 473 – 477.
Holmes, J.L., Kirkwood, C.D., Gerna, G., Clemens, J.D., Rao, M.R.,
Naficy, A.B., Abu-Elyazeed, R., Savarino, S.J., Glass, R.I., Gentsch,
J.R., 1999. Characterization of unusual G8 rotavirus strains isolated
from Egyptian children. Arch. Virol. 144, 1381 – 1396.
Hoshino, Y., Wyatt, R.G., Greenberg, H.B., Flores, J., Kapikian, A.Z.,
1984. Serotypic similarity and diversity of rotaviruses of mammalian
and avian origin as studied by plaque-reduction neutralization. J. Infect.
Dis. 149, 694 – 702.
Iturriza-Gomara, M., Anderton, E., Kang, G., Gallimore, C., Phillips, W.,
Desselberger, U., Gray, J., 2003. Evidence for genetic linkage between
the gene segments encoding NSP4 and VP6 proteins in common and
reassortant human rotavirus strains. J. Clin. Microbiol. 41, 3566 – 3573.
Kang, G., Green, J., Gallimore, C.I., Brown, D.W., 2002. Molecular epi-
demiology of rotaviral infection in South Indian children with acute
diarrhea from 1995 – 1996 to 1998 – 1999. J. Med. Virol. 67, 101 – 105.
Kapikian, A.Z., Hoshino, Y., Chanock, R.M., Perez-Schael, I., 1996. Effi-
cacy of a quadrivalent rhesus rotavirus-based human rotavirus vaccine
aimed at preventing severe rotavirus diarrhea in infants and young
children. J. Infect. Dis. 174, 65 – 72.
Kumar, S., Tamura, K., Jakobsen, I.B., Nei, M., 2001. MEGA2: molec-
ular evolutionary genetics analysis software. Bioinformatics 17,
1244 – 1245.
Li, B., Clark, H.F., Gouvea, V., 1994. Amino acid sequence similarity of
the VP7 protein of human rotavirus HCR3 to that of canine and feline
rotaviruses. J. Gen. Virol. 75, 215 – 219.
Liprandi, F., Gerder, M., Bastidas, Z., Lopez, J.A., Pujol, F.H., Ludert, J.E.,
Joelsson, D.B., Ciarlet, M.A., 2003. Novel type of VP4 carried by a
porcine rotavirus strain. Virology 315, 373 – 380.
17
Martella, V., Ciarlet, M., Camarda, A., Pratelli, A., Tempesta, M., Greco,
G., Cavalli, A., Elia, G., Decaro, N., Terio, V., Bozzo, G., Camero, M.,
Buonavoglia, C., 2003. Molecular characterization of the VP4, VP6,
VP7, and NSP4 genes of lapine rotaviruses identified in Italy: emer-
gence of a novel VP4 genotype. Virology 314, 358 – 370.
Mphahlele, M.J., Steele, A.D., 1995. Relative frequency of human rotavi-
rus VP4 (P) genotypes recovered over a ten-year period from South
African children with diarrhea. J. Med. Virol. 47, 1 – 5.
Nakagomi, T., Nakagomi, O., 2000. Human rotavirus HCR3 possesses a
genomic RNA constellation indistinguishable from that of feline and
canine rotaviruses. Arch. Virol. 145, 2403 – 2409.
Nakagomi, O., Mochizuki, M., Aboudy, Y., Shif, I., Silberstein, I., Naka-
gomi, T., 1992. Hemagglutination by a human rotavirus isolate as ev-
idence for transmission of animal rotaviruses to humans. J. Clin.
Microbiol. 30, 1011 – 1013.
Nakagomi, T., Tsunemitsu, H., Imagawa, H., Nakagomi, O., 2003. Ge-
nomic RNA constellation of recently emerging serotype G14 equine
rotavirus strains in Japan that is highly homologous with prototype
G3 and G14 strains previously identified in the United States of Amer-
ica. Arch. Virol. 148, 925 – 935.
Nicholas, K.B., Nicholas, H.B., Deerfield, D.W., 1997. GeneDoc: analysis
and visualization of genetic variation. Embnet News 4, 14.
Okada, J., Urasawa, T., Kobayashi, N., Taniguchi, K., Hasegawa, A., Mise,
K., Urasawa, S., 2000. New P serotype of group A human rotavirus
closely related to that of a porcine rotavirus. J. Med. Virol. 60, 63 – 69.
Palombo, E.A., 2002. Genetic analysis of Group A rotaviruses: evi-
dence for interspecies transmission of rotavirus genes. Virus Genes
24, 11 – 20.
Palombo, E.A., Bishop, R.F., 1995. Genetic and antigenic characterization
of a serotype G6 human rotavirus isolated in Melbourne, Australia.
J. Med. Virol. 47, 348 – 354.
Parashar, U.D., Hummelman, E.G., Bresee, J.S., Miller, M.A., Glass, R.I.,
2003. Global illness and deaths caused by rotavirus disease in children.
Emerging Infect. Dis. 9, 565 – 572.
Rao, C.D., Gowda, K., Reddy, B.S., 2000. Sequence analysis of VP4 and
VP7 genes of nontypeable strains identifies a new pair of outer capsid
proteins representing novel P and G genotypes in bovine rotaviruses.
Virology 276, 104 – 113.
Santos, N., Volotao, E.M., Soares, C.C., Albuquerque, M.C., da Silva, F.M.,
de Carvalho, T.R., Pereira, C.F., Chizhikov, V., Hoshino, Y., 2001. Ro-
tavirus strains bearing genotype G9 or P[9] recovered from Brazilian
children with diarrhea from 1997 to 1999. J. Clin. Microbiol. 39,
1157 – 1160.
Sato, K., Inaba, Y., Miura, Y., Tokuhisa, S., Matumoto, M., 1982. Isolation
of lapine rotavirus in cell cultures. Arch. Virol. 71, 267 – 271.
Sereno, M.M., Gorziglia, M.I., 1994. The outer capsid protein VP4 of
murine rotavirus strain Eb represents a tentative new P type. Virology
199, 500 – 504.
Thompson, J.D., Gibson, T.J., Plewniak, F., Jeanmougin, F., Higgins, D.G.,
1997. The ClustalX windows interface: flexible strategies for multiple
sequence alignment aided by quality analysis tools. Nucleic Acids Res.
24, 4876 – 4882.
Thouless, M.E., DiGiacomo, R.F., Neuman, D.S., 1986. Isolation of two
lapine rotaviruses: characterization of their subgroup, serotype and
RNA electropherotypes. Arch. Virol. 89, 161 – 170.
Urasawa, T., Taniguchi, K., Kobayashi, N., Mise, K., Hasegawa, A.,
Yamazi, Y., Urasawa, S., 1993. Nucleotide sequence of VP4 and VP7
genes of a unique human rotavirus strain Mc35 with subgroup I and
serotype 10 specificity. Virology 195, 766 – 771.
Wen, L., Nakayama, M., Yamanishi, Y., Nishio, O., Fang, Z.Y., Nakagomi,
O., Araki, K., Nishimura, S., Hasegawa, A., Muller, W.E., Ushijima, H.,
1997. Genetic variation in the VP7 gene of human rotavirus serotype 3
(G3 type) isolated in China and Japan. Arch. Virol. 142, 1481 – 1489.