Virology 380 (2008) 344353

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Virology
j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / y v i r o

Whole genome sequence and phylogenetic analyses reveal human rotavirus G3P[3]
strains Ro1845 and HCR3A are examples of direct virion transmission of canine/feline
rotaviruses to humans
Takeshi Tsugawa, Yasutaka Hoshino
Epidemiology Section, Laboratory of Infectious Disease, National Institute of Allergy and Infectious Disease, National Institutes of Health, Building 50, Room 6308, 50 South Drive,
MSC 8026, Bethesda, MD 20892-8026, USA

a r t i c l e

i n f o

Article history:
Received 10 June 2008
Returned to author for revision 3 July 2008
Accepted 31 July 2008
Available online 11 September 2008
Keywords:
Rotavirus
Interspecies transmission
Human rotavirus
Canine rotavirus
Feline rotavirus
Sequence analysis

a b s t r a c t
Rotaviruses, the major causative agents of infantile diarrhea worldwide, are, in general, highly speciesspecic. Interspecies virus transmission is thought to be one of the important contributors involved in the
evolution and diversity of rotaviruses in nature. Human rotavirus (HRV) G3P[3] strains Ro1845 and HCR3A
have been reported to be closely related genetically to certain canine and feline rotaviruses (RVs). Whole
genome sequence and phylogenetic analyses of each of these 2 HRVs as well as 3 canine RVs (CU-1, K9 and
A79-10, each with G3P[3] specicity) and 2 feline RVs (Cat97 with G3P[3] specicity and Cat2 with G3P[9]
specicity) revealed that (i) each of 11 genes of the Ro1845 and HCR3A was of canine/feline origin; (ii) canine
and feline rotaviruses with G3P[3] specicity bore highly conserved species-specic genomes; and (iii) the
Cat2 strain may have evolved via multiple reassortment events involving canine, feline, human and bovine
rotaviruses.
Published by Elsevier Inc.

Introduction
Group A rotaviruses remain the single most important etiologic
agents of severe diarrhea in infants and young children worldwide,
and the development and implementation of a safe and efcacious
rotavirus vaccine has been a global public health goal (Estes and
Kapikian, 2007). Rotaviruses are nonenveloped, icosahedral viruses of
the family Reoviridae with a genome consisting of 11 segments of
double-stranded RNA, each of which encodes one of six structural
(VP14, VP6 and 7) or six non-structural viral proteins (NSP16). The
outer capsid proteins VP7 (which denes G types) and VP4 (which
denes P types) evoke neutralizing and protective antibodies
independently (Greenberg et al., 1983; Hoshino et al., 1985; Oft
and Blavat, 1986). The segmented nature of the rotavirus genome
provides an opportunity for genomic reassortment, which, along with
point mutations, genome recombination and interspecies transmission, have been proposed to play an important role in the evolution
and diversity of rotaviruses in nature (Gouvea and Brantly, 1995;
Iturriza-Gmara et al., 2001; Maunula and von Bonsdorff, 2002;
Gentsch et al., 2005).
Rotaviruses are ubiquitous in a wide variety of mammalian and
avian species and, in general, highly species-specic. In addition, the
Corresponding author. Fax: +1 301 480 1387.
E-mail address: thoshino@niaid.nih.gov (Y. Hoshino).
0042-6822/$ see front matter. Published by Elsevier Inc.
doi:10.1016/j.virol.2008.07.041

genome RNA homology among rotavirus strains in a given animal

species has been reported to be much higher than that of rotavirus
strains derived from different animal species as demonstrated by
RNARNA hybridization (Flores et al., 1986). However, there are a
number of reports of atypical rotavirus strains isolated from humans
and other animal species that share genetic and antigenetic features of
virus strains derived from heterologous species (Palombo, 2002).
Fifteen G types have been established thus far (Rao et al., 2000; Estes
and Kapikian, 2007). Among them, G3 genotype is unique since it has
the broadest host range: G3 strains have been isolated from humans,
monkeys, horses, cows, pigs, sheep, goats, dogs, cats, rabbits and mice
(El-Attar et al., 2002; Varshney et al., 2002; Lee et al., 2003; Estes and
Kapikian, 2007). Indeed the VP7 gene sequence heterogeneity
observed among the G3 viruses (Nishikawa et al., 1989; Wen et al.,
1997) reects this host species heterogeneity. Moreover, G3 genotype
is the only G type that exists in certain animal species including dogs,
cats, rabbits and mice.
Human rotavirus (HRV) Ro1845 strain was originally isolated in 1985
from an infant with diarrhea in Israel (Aboudy et al., 1988) and HRV
HCR3A strain was recovered in 1984 from an asymptomatic infant in USA
(Li et al., 1993). Early on, certain unusual characteristics of the Ro1845 and
HCR3A strains were documented: both strains had a HA activity,
subgroup I specicity, a long electropherotype and a G3P[3] specicity,
characteristics which are typically found in animal rotaviruses (Aboudy et
al., 1988; Mackow et al., 1988; Nakagomi et al., 1992; Li et al., 1993;

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345

Table 1
Characteristics of rotavirus strains used in this study
Strain

Host

Diarrhea

Electropherotype

VP7 and VP4

genotype

Country
of origin

Year

Reference

Ro1845
HCR3A
CU-1
K9
A79-10
Cat97
Cat2

Human
Human
Dog
Dog
Dog
Cat
Cat

+
NA
+

Long
Long
Long
Long
Long
Long
Long

G3P[3]
G3P[3]
G3P[3]
G3P[3]
G3P[3]
G3P[3]
G3P[9]

Israel
USA
USA
USA
USA
Australia
Australia

1985
1984
1980
1979
1979
1984
1984

Aboudy et al. (1988)

Li et al. (1993)
Hoshino et al. (1982)
Fulton et al. (1981)
Hoshino et al. (1983)
Birch et al. (1985)
Birch et al. (1985)

NA: not available

Taniguchi et al., 1994; Martella et al., 2001a,b; Lee et al., 2003). RNARNA
hybridization assays showed that each of these 2 human G3P[3] strains
formed 10 or 11 hybrid bands against selected feline or canine strains,
which suggested that these strains were of possible canine/feline origin
(Nakagomi et al., 1990, Nakagomi and Nakagomi, 2000). Previously, the
VP4 gene of strain Ro1845 and both the VP4 and VP7 genes of strain
HCR3A were sequenced and analyzed (Li et al., 1993, 1994; Nakagomi et
al., 1993; Taniguchi et al., 1994). In the current study, we sequenced and
analyzed all eleven genes of each of these 2 HRV G3P[3] strains as well as
3 canine (CRV CU-1, K9 and A7910, each with G3P[3] specicity) and 2
feline (FRV Cat97 with G3P[3] specicity and Cat2 with G3P[9] specicity)
rotavirus strains in an attempt to (i) further characterize these 2 human
strains relative to canine and feline strains at a molecular level with
special emphasis on a possible scenario of animal to human interspecies
transmission; and (ii) analyze the extent of phylogenetic distinctness of
each of 11 canine and feline rotavirus genes.
Results
Comparative analyses of whole genome sequences as visualized by
mVISTA module
Table 1 summarizes rotavirus strains used in this study. By using the
mVISTA module (http://genome.lbl.gov/vista/index.shtml) (Frazer et al.,
2004), we aligned whole genome nucleotide sequences of (i) the 6
rotavirus strains analyzed in the present study and in addition, 3 strains
AU-1 (HRV), DS-1 (HRV) and simian rotavirus RRV against the CRV CU-1
strain (Fig. 1A); and (ii) strains Cat97 (FRV), AU-1, DS-1, Wa (HRV) and
bovine rotavirus RF strain against the FRV Cat2 strain (Fig. 1B). As
visualized in Fig. 1A, whole genomes of all 6 G3P[3] strains in this study
were highly conserved (9499% nt) suggesting that the 2 G3P[3] human
strains Ro1845 and HCR3A were of canine/feline origin. Only genes
encoding VP1, VP2, VP3 and NSP4 of the Cat2 strain shared a high degree
of sequence conservation with the 6 G3P[3] strains (9598% nt). Although
the VP6 gene of the AU-1 strain, the VP2 gene of the DS-1 strain and the
NSP5 gene of the RRV strain shared a relatively high degree of sequence
conservation with the corresponding genes of the 6 G3P[3] strains, the
magnitude of conservation in each of these three genes among the 6G3P
[3] strains was much higher (Table 2). The Cat2 strain demonstrated a
high degree sequence conservation in (i) genes encoding VP4, VP6, VP7,
and NSP1 with AU-1 strain (9195% nt); (ii) VP2 gene with DS-1 strain
(91%); (iii) NSP2 gene with Wa strain (92%); and (iv) NSP3 and NSP5 genes
with RF strain (92 and 97% nt) (Table 2).
Sequence and phylogenetic analyses of structural viral proteins
Core proteins VP1, VP2 and VP3
Each of three cognate genes encoding VP1, VP2 and VP3 proteins,
respectively, of the 7 strains analyzed shared a high sequence identity
(9598% nt and 97100% aa) and formed a distinct phylogenetic genotype
cluster (Figs. 2AC). Based on a novel phylogenetic genotype classication
system proposed recently by Matthijnssens et al. (2008), genes encoding
VP1, VP2 and VP3 of each of the 7 strains were shown to belong to

genotypes R3 (VP1), C2 (VP2) and M3 (VP3), respectively (Table 2). The

core proteins of the 7 strains displayed conservation of the 4 RNAdependent-RNA-polymerase motifs in VP1 (Kamer and Argos, 1984; Poch
et al., 1989; Ito et al., 2001); the 5 heptad repeats of basic and acidic
residues (CU-1 and K9 with only one amino acid substitution); two
leucine zippers in VP2 (Kumar et al., 1989; Mitchell and Both, 1990); and
KXTAMDXEXP (a possible casein kinase II phosphorylation site) and
KXXGNNH motifs in VP3 (Cook and McCrae, 2004).
Intermediate capsid protein VP6
All 6 G3P[3] strains shared a high VP6 identity (9699% nt and 99
100% aa) whereas the Cat2 strain exhibited moderate to high VP6
identities (8788% nt and 9798% aa) with the 6 G3P[3] strains (Figs.
1A and 2E). Based on the proposed nucleotide identity cut-off value of
80% (Matthijnssens et al., 2008), the VP6 gene of all 7 strains analyzed
belonged to the I3 genotype (Table 2). In the region associated with
subgroup specicity, all seven strains had conserved residues at 305Ala, 310-Asn, 339-Asn, 342-Leu and 348-Ser (except for the Cat2 strain
with 342-Met) which indicated mixed characteristics of genogroup I
and II rotaviruses (Iturriza-Gmara et al., 2002; Lpez et al., 1994; Tang
et al., 1997).
Outer capsid proteins VP4 and VP7
All 6 G3P[3] strains shared a high VP4 sequence identity (9598%
nt and 9798% aa) (Fig. 1A) and with other G3P[3] strains FRV FRV64
(9598% nt and 9798% aa) and CRV RV198/95(VP8: 9396% nt and
9698% aa) available in GenBank, and together formed a phylogenetic
cluster distinct from other P[3] clusters (Fig. 2D). The VP4 of FRV Cat2
strain, which displayed a low sequence identity (6768% nt and 68
69% aa) with those of the 6 G3P[3] strains (Fig. 1A), was found to be
closely related to the VP4 of strains HRV AU-1 (G3P[9]), K8 (G1P[9])
and FRV FRV-1 (G3P[9]) (9596% nt and 9798% aa) which formed a
distinct phylogenetic P[9] cluster (Fig. 2D). Each of the 7 VP4 proteins
analyzed bore 2 or 3 conserved trypsin cleavage sites (Lpez et al.,
1985; Arias et al., 1996), conserved cysteine residues (Patton et al.,
1993a,b), and the 21 integrin ligand site DGE (Coulson et al., 1997).
Although all 7 strains were classied genotype G3, the Cat2 strain
displayed only a moderately high VP7 identity (7980% nt and 91% aa)
with the 6 G3P[3] strains, which among them shared a high sequence
identity (9598% nt and 99100% aa) (Fig. 1A). The VP7s of the 6 G3P
[3] strains clustered together phylogenetically to form G3a whereas
that of the Cat2 strain clustered together with AU-1-like VP7s to form
G3b (Fig. 2F) (Matthijnssens et al., 2008). The VP7 antigenic regions A,
B, C and F of all 6 G3P[3] strains were conserved completely whereas
the Cat2 VP7 had mutation(s) in regions B (1/6), C (2/13) and F (2/8)
comparing to the 6 G3P[3] strains (Dyall-Smith et al., 1986; Kirkwood
et al., 1993; Ciarlet et al., 1997).
Sequence and phylogenetic analyses of non-structural proteins
NSP1
All 6 G3P[3] strains shared a high NSP1 sequence identity (9497%
nt and 9497% aa) and formed one phylogenetic NSP1 cluster (Figs. 1A

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T. Tsugawa, Y. Hoshino / Virology 380 (2008) 344353

Fig. 1. mVISTA whole genome nucleotide alignment comparing (A) the canine CU-1 strain with 6 other rotavirus strains (K9, A79-10, Ro1845, HCR3A, Cat97 and Cat2) of human,
canine or feline origin analyzed in the present study, and 3 prototype strains (AU-1, DS-1 and RRV) which constitute a genotype composition of each of the 6 G3P[3] strains (CU-1, K9,
A79-10, Ro1845, HCR3A and Cat97); and (B) the feline Cat2 strain with Cat97 strain and 4 prototype strains (AU-1, DS-1, Wa and RF) which constitute a genotype composition of the
Cat 2 strain. Percentage (right y-axis) indicates sequence identity, and shading indicates level of conservation.

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347

Table 2
Comparison of nucleotide identity (%) of 11 genome segments of seven rotavirus strains (Ro1845, HCR3A, CU-1, K9, A79-10, Cat97 and Cat2) analyzed in the present study
Gene
encoding

VP1
VP2
VP3
VP4
VP6
VP7
NSP1
NSP2
NSP3
NSP4
NSP5
a
b
c
d

% nt
cut-off
valuea

83
84
81
80
85
80
79
85
85
85
91

Genotypea
of 6 G3P
[3] strainsb

R3
C2
M3
P[3]
I3
G3a
A9
N2d
T3
E3
H6

% nt identity of 6 G3P[3] strains

against indicated strain(s)

Genotype
of Cat2
straina

(Prototype genotype
strainc)

6 G3P[3] strains

8687 (AU-1)
9091 (DS-1)
8384 (AU-1)
8081 (RRV)
8889 (AU-1)
8485 (RRV)
8384 (RRV)
8384d (DS-1)
8687 (AU-1)
8788 (AU-1)
9293 (RRV)

9598
9698
9597
9598
9699
9598
9497
9698
9697
9598
9699

R3
C2
M3
P[9]
I3
G3b
A3
N1
T6
E3
H3

% nt identity of Cat2 strain

against indicated strain(s)
(Prototype genotype
strainc)

(strain(s) exhibiting
highest identity vs Cat2)

87 (AU-1)
91 (DS-1)
82 (AU-1)
95 (AU-1)
92 (AU-1)
91 (AU-1)
93 (AU-1)
92 (Wa)
92 (RF)
88 (AU-1)
93 (AU-1)

9596 (6 G3P[3])
9698 (6 G3P[3])
96 (6 G3P[3])
96 (K8)
92 (AU-1)
97 (KC814)
93 (AU-1)
95 (Dhaka25-02)
92 (RF)
98 (FRV70)
97 (RF)

Percentage nucleotide cut-off values and genotypes proposed by Matthijnssens et al. (2008).
Six G3P[3] strains: Ro1845, HCR3A, CU-1, K9, A79-10 and Cat97.
Prototype genotype strains used by Matthijnssens et al. (2008).
nt identity versus DS-1 is 8384%, however, nt identity versus DRC88, DRC86 and UK is 85%.

and 3A). The FRV Cat2 strain, which displayed low sequence identity
(2526% nt and 36% aa) to the 6 G3P[3] strains, clustered with the HRV
AU-1 strain (Figs. 1A and 3A). Based on the proposed 79% nucleotide
identity cut-off value (Matthijnssens et al., 2008), the NSP1 gene of the
6 G3P[3] strains and that of the Cat2 strain belonged to genotypes A9
and A3, respectively (Table 2). Each of the 7 NSP1 proteins retained
conserved cysteine-rich putative zinc nger domains (Hua et al., 1993)
and proline residues (Xu et al., 1994).
NSP2 and NSP3
All 6 G3P[3] strains shared a high NSP2 or NSP3 sequence identity
(9698% nt and 97100% aa), and formed a distinct NSP2 or NSP3
phylogenetic cluster (Figs. 1A and 3BC). The Cat2 strain displayed
moderately high nucleotide (NSP2; 8283% and NSP3; 7879%,
respectively) and amino acid (NSP2; 8990% and NSP3; 82%,
respectively) identities with the 6 G3P[3] strains (Fig. 1A). In the
phylogenetic analysis, the Cat2 strain was closely related to the HRV
strains Dhaka25-02, RMC321 and Wa in the NSP2 gene (9295% nt)
(Fig. 3B), and to the bovine rotavirus RF and HRV B4106 strains in the
NSP3 gene (8992% nt) (Fig. 3C). The NSP2 genes of the 6 G3P[3]
strains shared a nucleotide identity of 8384% with the HRV DS-1
strain, and 85% with other DS-1 genotype viruses including HRV
DRC88, DRC86 as well as bovine rotavirus UK strains. Thus, based on
the proposed 85% nucleotide identity cut-off value (Matthijnssens et
al., 2008), the NSP2 genes of the 6 G3P[3] strains and that of the Cat2
strain were assigned to genotypes N2 and N1, respectively. The NSP3
genes of 6 G3P[3] strains and that of the Cat2 strain belonged to
genotypes T3 and T6, respectively (Table 2). The putative NSP2 RNAbinding domain residues were highly conserved, and 4 cysteines
residues (6, 8, 85 and 285 aa) were completely conserved in all 7
strains (Patton et al., 1993b). In NSP3, the consensus sequence (I/L)
xxM(I/L)(S/T)xxG, short acidic region, hydrophobic heptad repeat
region and cysteines residues (123, 139 and 306 aa) were also
conserved (Mattion et al., 1992; Rao et al., 1995).
NSP4
All 7 strains shared a high NSP4 sequence identity (9598% nt and
9698% aa) and clustered together phylogenetically with other
selected feline strains (FRV70 and FRV64) to form NSP4 genogroup C
(Figs. 1A and 3D)(Ciarlet et al., 2000). Based on the proposed
nucleotide identity cut-off value of 85% (Matthijnssens et al., 2008),
the NSP4 genes of the 7 strains belonged to genotype E3 (Table 2). In
each of the 7 NSP4 proteins, the 3 conserved hydrophobic domains
(Chan et al., 1988) and the 2 conserved N-glycosylation sites (Both et
al., 1983) were almost identical, with the Ro1845 and A79-10 strains

bearing only one amino acid substitution. The domain associated with
the formation of NSP4 homotetramers (Taylor et al., 1996), the
enterotoxic and membrane destabilizing peptide domain (Ball et al.,
1996, Tian et al., 1996), the VP4 binding domain (Au et al., 1993), and
the single-shelled (ss) particle-binding domain (Au et al., 1993) were
highly conserved among all 7 strains.
NSP5
The NSP5 genes of all 6 G3P[3] strains shared a high sequence
identity (9699% nt and 96100% aa) and clustered together
phylogenetically (Figs. 1A and 3E). The Cat2 strain, which displayed
a moderately high NSP5 identity (8890% nt and 9092% aa) with the
6 G3P[3] strains (Fig. 1A), was shown to be closely related to bovine
rotavirus RF strain (97% nt) (Figs. 1B and 3E). Based on the proposed
91% nucleotide identity cut-off value (Matthijnssens et al., 2008), the
NSP5 genes of the 6 G3P[3] strains and that of the Cat 2 strain
belonged to genotypes H6 and H3, respectively (Table 2). The SDSAS
motif associated with NSP5 hyperphosphorylation (Eichwald et al.,
2004) and the 2 DxDxD motifs related to the formation of viroplasmlike structures (Sen et al., 2007) were almost identical among all 7
strains, with the Cat2 strain bearing only one amino acid substitution.
Discussion
Rotaviruses are, in general, highly species-specic, however, an
increasing number of reports describe the detection of atypical
rotaviruses from humans and other animal species (Isegawa et al., 1992;
Li et al., 1993; Nakagomi et al., 1993; Palombo, 2002; Laird et al., 2003;
Khamrin et al., 2006; Matthijnssens et al., 2006, 2008; Banerjee et al.,
2007; De Grazia et al., 2007). Rotavirus strains bearing a G3P[3] specicity
are typically found in animal host species including dogs, cats and
monkeys (Mackow et al., 1988; Taniguchi et al., 1994; Martella et al.,
2001a,b; Estes and Kapikian, 2007). In addition, such strains have also
been detected in goats and cows (Lee et al., 2003; Ghosh et al., 2007).
Although the published data regarding canine rotavirus G and P types are
limited, reports from the USA, Japan, Italy and Korea (Hoshino et al., 1983;
Nakagomi et al., 1989; Gouvea et al., 1994; Taniguchi et al., 1994; Martella
et al., 2001a; Kang et al., 2007) indicate that all the canine rotavirus strains
analyzed thus far bear G3P[3] specicity. Similarly, based on a limited
number of reports from the USA, Japan and Australia (Birch et al., 1985;
Gouvea et al., 1994; Taniguchi et al., 1994; Mochizuki et al., 1997), all the
feline rotavirus strains display either G3P[3] or G3P[9] specicity. In
addition, one seroepidemiological study performed in Bochum, Germany
during 19911992 season reported that 91% of the 22 cat sera had
neutralizing antibodies to G3 viruses (strains AU223 and SA11) but no

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T. Tsugawa, Y. Hoshino / Virology 380 (2008) 344353

Fig. 2. Bootstrap consensus cladograms for the nucleotide sequences of VP1, VP2, VP3, VP4, VP6 and VP7 (AF). Phylogenetic analysis was performed using the maximum likelihood
method provided by the DNAML program of PHYLIP 3.67. Bootstrap values (1000 replicates) are shown. Avian rotavirus PO-13 strain was used as an outgroup. Arrows and arrowhead
indicate the 6 G3P[3] strains and Cat2 strain, respectively, analyzed in this study; human Ro1845 and HCR3A strains are boxed. Designation of species of origin: Hu: human; Ca:
canine; Fe: feline; Si: simian; Bo: bovine; Cap: caprine; Av: avian. Nucleotide accession numbers collected from GenBank: VP1(A); Po-13, AB009629; DS-1, DQ870505; Wa,
DQ490539; AU-1, DQ490533; SA11[H96], DQ838640; B4106, AY740741; RF, J04346; RRV, EF583006. VP2(B); Po-13, AB009630; DS-1, DQ870506; AU-1, DQ490536; SA11[H96],
DQ838635; B4106, AY740740; RF, X14057; Wa, X14942; RRV, EF583007. VP3 (C); Po-13, AB009631; AU-1, DQ490537; SA11[H96], DQ838645; B4106, AY740739; DS-1, AY277914; Wa,
AY267335; RF, AY116592; RRV, EF583008. VP4(D); Po-13, AB009632; FRV64, D14723; RV52/96, AF339844; RV198/95, AF339843; PA260/97, EF442735; K8, D90260; FRV-1, D10971;
02-92, AB008289; AU-1, D10970; CMH222, DQ288661; GRV, AB055967; CRI33594, EF202611; RRV, M18736. VP6(E); Po-13, D16329; DS-1, DQ870507; AU-1, DQ490538; SA11[H96],
DQ838650; CMH222, DQ288659; B4106, AY740737; RF, K02254; Wa, K02086; PA260/97, EF442738; RS52/96, EF442739; CRI33594, EF175920; RV198/95, EF442740; RRV, EF583009.
VP7(F); Po-13, D82979; SA11[H96], DQ838620; CMH222, AY707792; B4106, AY456382; AU-1, D86271; KC814, AJ311739; CC425, AJ311738; RV52/96, AF271090; RV198/95, AF271089;
GRV, AB056650; CMH120/04, DQ923797; PA260/97, EF442733; 71771042, AJ488587; CRI33594, EF175919; RRV, M21650.

antibodies neutralizing G1 (strain Wa), G2 (strain S2) and G4 (strain

Hochi) viruses (Streckert et al., 1993). Thus, these studies have
demonstrated that rotaviruses bearing G3P[3] specicity are common
in both dogs and cats. Interestingly, there have been at least 6 reports
describing the detection of G3P[3] strains in humans: Ro1845 (Aboudy et

al., 1988), HCR3A and HCR3B (Li et al., 1993; Santos et al., 1998), CMH222
(Khamrin et al., 2006), PA260/97 (De Grazia et al., 2007) and CRI33594
(Banerjee et al., 2007). Among them, the Ro1845 and HCR3A strains,
which were isolated in the mid-1980s, were analyzed fairly extensively
with various assays (e.g., RNARNA hybridization, restriction fragment

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349

Fig. 3. Bootstrap consensus cladograms for the nucleotide sequences of NSP1, NSP2, NSP3, NSP4 and NSP5 (AE). Phylogenetic analysis was performed using the maximum likelihood
method provided by the DNAML program of PHYLIP 3.67. Bootstrap values (1000 replicates) are shown. Avian rotavirus PO-13 strain was used as an outgroup. Arrows and arrowhead
indicate the 6 G3P[3] strains and Cat2 strain, respectively, analyzed in this study; human Ro1845 and HCR3A strains are boxed. Panel D (NSP4 gene): asterisk indicates NSP4 genotype
designations proposed by Matthijnssens et al. (2008) followed by those used previously in parentheses. Designation of species of origin: Hu: human; Ca: canine; Fe: feline; Si: simian;
Bo: bovine; Cap: caprine; Av: avian. Nucleotide accession numbers collected from GenBank: NSP1(A); Po-13, AB009633; SA11[H96], DQ838599; B4106, AY740735; AU-1, D45244;
FRV64, D78362; Wa, L18943; DS-1, L18945; RF, M22308; RRV, U08433. NSP2(B); Po-13, AB009625; AU-1, DQ490534; SA11[H96], DQ838615; Dhaka25-02, DQ146656; B4106,
AY740734; RF, Z21640; RMC321, AF506293; Wa, L04534; DS-1, L04529; DRC86, DQ005118; DRC88, DQ005107; UK, J02420. NSP3(C); Po-13, AB009626; AU-1, DQ490535; SA11[H96],
DQ838610; DS-1, EF136660; B4106, AY740733; Wa, X81434; RF, Z21639; RRV, AY065842. NSP4(D); Po-13, AB009627; SA11[H96], DQ838625; Wa, AF200224; RS15, D88832; FRV64,
D88833; FRV-1, D89874; FRV70, AB048196; AU-1, D89873; GRV, AB055968; CMH222, DQ288660; DS-1, AF174305; B4106, AY740732; RF, AY116593; CRI33594, EF175921; RRV,
L41247. NSP5(E); Po-13, AB009628; Wa, AF306494; DS-1, M33608; SA11[H96], DQ838630; AU-1, AB008656; T152, DQ146706; B4106, AY740731; RF, AF188126; RRV, AF306492.

length polymorphism assay and sequencing of VP4 and VP7 genes)

(Nakagomi et al., 1990, 1993; Nakagomi and Nakagomi, 2000; Li et al.,
1993, 1994; Vonsover et al., 1993; Taniguchi et al., 1994; Santos et al.,
1998). RNARNA hybridization assays, which demonstrated that each of
these two human G3P[3] strains formed 10 or 11 hybrid bands versus
certain canine and feline rotavirus strains bearing a G3P[3] specicity,
suggested a possible transmission of whole rotavirus virions of canine/

feline origin to humans (Nakagomi et al., 1990; Nakagomi and Nakagomi,

2000). In the present study, we sequenced and analyzed all 11 genes of
strains Ro1845 and HCR3A as well as 3 canine (CU-1, K9 and A79-10, each
with G3P[3] specicity) and 2 feline (Cat97 with G3P[3] specicity and
Cat2 with G3P[9] specicity) rotavirus strains, and demonstrated that (i)
these 2 human G3P[3] strains were indeed examples of whole virion
transmission cases of canine/feline rotaviruses to humans; and (ii) each of

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T. Tsugawa, Y. Hoshino / Virology 380 (2008) 344353

11 cognate genes of the 6 G3P[3] strains shared a characteristically high

nucleotide identity and formed a distinct phylogenetic cluster suggesting
that canine and feline rotaviruses with G3P[3] specicity bore highly
conserved species-specic genomes that were distinct from genomes of
human, simian, bovine, porcine or avian rotavirus origin.
Previous analyses of selected CRVs, FRVs and HRVs by RNARNA
hybridization and hemagglutination assays (Nakagomi et al., 1990;
Nakagomi and Nakagomi, 1991; Mochizuki et al., 1992, 1997)
demonstrated the existence of 2 genogroups among them: one a
caninefeline group (e.g., CRV strains CU-1 and K9, and FRV strains
Cat97 and FRV64), and the other a humanfeline group (e.g., HRV
strain AU-1 and FRV strain FRV-1). In addition, the AU-1 (G3P[9])
strain was shown to be the rst example of a suspected interspecies
transmission event from a cat to a human (Nakagomi and Nakagomi,
1989). Sequence analyses of each of 11 cognate genes of the 6 G3P[3]
strains conrmed the existence of 2 such genogroups (a caninefeline
group: strains CU-1, K9, A79-10 and Cat97; and a humanfeline group:
strain AU-1) (Figs. 1A, 2 and 3).
Previously, analysis by RNARNA hybridization assay suggested
that the FRV Cat2 strain was a reassortant virus between a canine
feline virus and a humanfeline virus because a K9 probe belonging to
a caninefeline group formed 5 hybrid bands and a FRV-1 probe
belonging to a humanfeline group formed 6 hybrid bands with the
Cat2 genome (Mochizuki et al., 1992). In the present study, we showed
that the Cat2 strain displayed the highest nucleotide identity in (i) the
VP1, VP2 and VP3 genes with the 6 G3P[3] strains; (ii) the VP4 gene
with HRV K8 (G1P[9]) strain; (iii) the VP6 and NSP1 genes with HRV
AU-1 (G3P[9]) strain; (iv) the VP7 gene with HRV KC814 (G3P[9])
strain; (v) the NSP2 gene with HRV Dhaka25-02 (G12P[8]) strain; (vi)
the NSP3 and NSP5 genes with bovine rotavirus RF (G6P[1]) strain;
and (vii) NSP4 gene with FRV FRV70 strain (Table 2). These ndings
suggest that the Cat2 strain may have evolved via multiple reassortment events involving canine, feline, human and bovine rotaviruses.
Previously, sequence and phylogenetic analyses of 4 genes encoding
VP4, VP6, VP7 and NSP4, respectively, of HRV CMH222 strain
suggested that the CMH222 strain was generated through multiple
reassortment events involving caprine, simian and human rotaviruses
(Khamrin et al., 2006).
Although each of 11 cognate genes of the 6 G3P[3] strains analyzed
in the present study displayed high sequence identity (9499% nt and
94100% aa), the clinical symptoms of the hosts from which these
strains were isolated varied depending upon the strain. The HRV
Ro1845, CRV K9 and FRV Cat97 strains were derived from diarrheal
hosts whereas the HRV HCR3A and CRV CU-1 strains were isolated
from asymptomatic hosts (Table 1). By using a genetic approach, we
(Hoshino et al., 1995) and others (Oft et al., 1986; Bridger et al., 1992;
Ball et al., 1996; Graff et al., 2002; Mossel and Ramig, 2002; Barro and
Patton, 2005) showed previously that at least 6 genome segments (i.e.,
genes encoding VP3, VP4, VP7, NSP1, NSP3 and NSP4) were involved in
pathogenesis of rotaviruses. Therefore, it was of interest to analyze the
involvement of these 6 virulence-associated genes in the observed
differences in clinical signs of the hosts by comparing whole genome
nucleotide sequences between 3 symptomatic (Ro1845, K9 and Cat97)
and 2 asymptomatic (HCR3A and CU-1) strains. Analyses revealed the
existence between the two groups of (i) 11 mutations in the VP7 gene,
(ii) 6 mutations in the VP3 gene, (iii) 4 mutations in the NSP4 gene, (iv)
2 mutations each in the VP1, VP4 and NSP3 genes, (v) 1 mutation each
in the VP2, NSP1 and NSP5 genes, and (vi) no mutations in the VP6 and
NSP2 genes. Although the numbers of symptomatic and asymptomatic infections were too few to draw a signicant association,
comparative whole genome sequence analyses were in agreement
with previous genetic studies, since there were several mutations in
the virulence-associated genes and a few mutations or no mutations
at all in the genes not associated with virulence.
The Ro1845 and HCR3A strains (which were shown to be of canine/
feline origin in this study) were recovered from a heterologous host

(i.e., humans) whereas the CU-1, K9, A79-10 and Cat97 strains were
recovered from a homologous host (i.e., dogs or cats) (Table 1). In
order to analyze whether the canine/feline rotaviruses acquired any
mutation(s) during the replication in a heterologous host, we
compared whole genome nucleotide sequences of the 4 strains (i.e.,
CU-1, K9, A79-10 and Cat97) recovered from a homologous host to
those of the 2 strains (i.e., Ro1845 and HCR3A) isolated from a
heterologous host. The following mutations were detected between
the 2 groups: (i) 2 each in the VP2, VP3 and NSP4 gene, (ii) 1 in the
NSP1 and NSP3 gene, and (iii) none in the VP1, VP4, VP6, VP7, NSP2
and NSP5 genes. Since no mutations were detected in the VP1, VP4,
VP6, VP7, NSP2 and NSP5 genes between the 2 groups, these proteins
did not appear to restrict human (heterologous) infection by canine/
feline G3P[3] viruses.
Based on a distinct nucleotide percentage identity cut-off value
for each of 11 segments of rotavirus, Matthijnssens et al. (2008)
recently proposed a novel full genome-based rotavirus classication and nomenclature system. They have identied four VP1 (R),
ve VP2 (C), six VP3 (M), twenty seven VP4 (P), eleven VP6 (I),
nineteen VP7 (G), fourteen NSP1 (A), ve NSP2 (N), seven NSP3
(T), eleven NSP4 (E) and six NSP5 (H) genotypes, respectively,
based on nucleotide identity cut-off values of 83%, 84%, 81%, 80%,
85%, 80%, 79%, 85%, 85%, 85% and 91%, respectively (Table 2). With
these proposed cut-off values, each of 11 cognate genome
segments of the 6 G3P[3] strains (i.e., Ro1845, HRC3, CU-1, K9,
A79-10 and Cat97) is classied as R3, C2, M3, P[3], I3, G3a, A9,
N2, T3, E3, and H6, respectively: genotype 3 (e.g., R3, M3, I3, etc)
represents HRV AU-1-like; genotype 2 (e.g., C2) represents HRV
DS-1-like; genotype 9 (e.g., A9) represents rhesus-lapine-K9
rotavirus-like; and genotype 6 (e.g., H6) represents RRV-like.
Similarly, each of 11 genome segments of the Cat2 strain is
classied as R3, C2, M3, P[9], I3, G3b, A3, N1, T6, E3, and H3,
respectively with genotype 1 (e.g., N1) representing HRV Wa-like
(Table 2). However, it has become clear from our whole genome
sequence analyses that each of 11 cognate genome segments of
the 6 G3P[3] viruses shares characteristically high nucleotide
identities and forms a distinct phylogenetic cluster. For example,
VP1 genes of the 6 G3P[3] strains share 8687% nucleotide
identities with the prototype genotype strain AU-1 (R3), however,
they share much higher nucleotide identities (9598%), which is
reected in the formation of a distinct cluster in the phylogenetic
tree. These ndings appear to indicate that canine/feline rotaviruses with a G3P[3] specicity bear a distinctly conserved
genome. In the proposed classication system, Matthijnssens et
al. (2008) divide the VP7 G3 genotype into at least 4 subgenotypes (i.e., G3a, G3b, G3-SA11, and G3-murine). Our data
suggest that a similar subgenotype classication (or gene allele
classication) system may also be needed for the remaining 10
rotavirus genome segments.
It is generally thought that rotavirus infections in dogs and cats are
milder than those in humans and farm animals (e.g., cows, horses and
pigs) (Hoskins, 1998; Harbour, 1998). In addition, the detection of
canine/feline-like viruses in humans is rare. However, since (i)
naturally-occurring caninehuman and felinehuman reassortant
rotaviruses have been detected (Mochizuki et al., 1992; Laird et al.,
2003); and (ii) a small outbreak of diarrhea among children caused by
feline-like P[9] viruses has been reported in Brazil (Santos et al., 2001),
rotavirus surveillance in pet animals including dogs and cats merits
further exploration.
Materials and methods
Viruses
HRV Ro1845 strain was isolated from a 3-week-old infant with
diarrhea in Israel and HRV HCR3A strain was recovered from an

T. Tsugawa, Y. Hoshino / Virology 380 (2008) 344353

asymptomatic infant (age unknown) who was a vaccinee in a phase I

clinical trial of the WC3 bovine rotavirus candidate vaccine in USA
(Aboudy et al., 1988; Li et al., 1993; Nakagomi et al., 1992). Canine
rotavirus strains CU-1, K9 and A79-10 were isolated in the USA, and
feline rotavirus strains Cat97 and Cat2 were isolated in Australia. Strains
K9 and Cat97 were derived from animals with diarrhea whereas strains
CU-1 and Cat2 were isolated from asymptomatic animals (Table 1).

351

rotavirus strains Ro1845, HCR3A, Cat97 and Cat2, K9 and AU-1,

respectively; K. Wollenberg for phylogenetic analyses of the genes,
and A. Z. Kapikian for continuing support. This work was supported by
the Intramural Research Program of the National Institute of Allergy
and Infectious Diseases, National Institutes of Health, USA. There is no
conict of interest to declare.
Appendix A. Supplementary data

RNA extraction and RT-PCR

Double-stranded RNA was extracted with TRIzol (Invitrogen) from
infected cell culture lysates as described previously (Santos et al., 2001).
The primers used for the RT-PCR were listed in the Supplemental Table S1.
Extracted double-stranded RNA (2 l) was added to 1 l of dimethyl
sulfoxide. The mixture was heated at 94 C for 3 min and placed on ice,
followed by the addition of 8 l of 1rst-strand buffer containing 25 mM
DTT; a 625 M concentration (each) of dATP, dCTP, dGTP, and dTTP; 40 U
of SuperScript II (Invitrogen); and a 1.25 M concentration (each) of
primers (see Table S1). RT was performed at 45 C for 45 min, followed by
94 C for 3 min. PCR was performed in 50 l of 1green GoTaq exi buffer
containing 1. 5 mM MgCl2, a 400 nM concentration of each primer, a
200 M concentration of each of the four deoxynucleoside triphosphates,
2.5 U of GoTaq DNA polymerase (Promega), and 3 l of the RT product.
Amplication was done under the following conditions: initial denaturation at 95 C for 15 min; 40 cycles at 94 C for 45 s, 50 C for 45 s, and 70 C
for 2.5 min; and a nal extension at 70 C for 7 min (Matthijnssens et al.,
2006). The PCR products were analyzed by electrophoresis in 1% agarose
gel in 1TrisacetateEDTA buffer containing 0. 2 g/ml of ethidium
bromide. RT-PCR was performed using specic primers (see Table S1)
designed from GenBank database or published data (Gouvea et al., 1990;
Kudo et al., 2001; Matthijnssens et al., 2006).
Nucleotide sequencing
The PCR amplicons were puried with the Wizard SV Gel and PCR
Clean-up system (Promega) and sequenced with the BigDye terminator v3.1 cycle sequencing reaction kit (Applied Biosystems Group)
on an ABI 3730 DNA analyzer (Applied Biosystems Group). Sequencing
primers are listed in Table S1 in the supplemental material.
Determination of the 5and 3 terminal sequence of VP4 genes
To obtain the complete ORF nucleotide sequence of the VP4 gene, 5
and 3 terminal sequences of the VP4 gene were determined using a fulllength amplication of cDNAs (FLAC) method with modication (Maan et
al., 2007). Specic primers used in FLAC were given in Table S1.
Sequence analysis
Sequence les were analyzed using Sequencher 4.7 (Gene Codes
Corporation) and MacVector 9.5.2 (Accelrys). Multiple sequence
alignments were prepared using ClustalW (EMBL-EBI) or mVISTA
(http://genome.lbl.gov/vista/index.shtml) (Frazer et al., 2004).
Searches for similar sequences were conducted using BLAST. Phylogenetic analysis was performed using DNAML (PHYLIP 3.67). The
nucleotide sequence data reported in this paper have been submitted
to the GenBank nucleotide sequence database and the accession
numbers are as follows: Ro1845, EU708890EU708900; HCR3A,
EU708901EU708911; CU-1, EU708912EU708922; K9, EU708923
EU708933; A79-10, EU708934EU708944; Cat97, EU708945
EU708955; and Cat2, EU708956EU708966.
Acknowledgments
We thank R. W. Jones for expert technical assistance; Y. Aboudy, H.
F. Clark, I. D. Gust, R. W. Fulton and O. Nakagomi for providing us with

Supplementary data associated with this article can be found, in

the online version, at doi:10.1016/j.virol.2008.07.041.
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