Three G9 samples (R143, R146, and R160) were randomly selected, and the
VP7-encoding gene of each virus was sequenced and compared to other G9
rotavirus VP7 gene sequences available in the GenBank. The best match
was with the American G9 strain US1205, which exhibited 99.0 to 99.5%
nucleotide and 98.6 to 99.1% deduced amino acid identity (Table
2). The phylogenetic analysis of the nucleotide sequences revealed the existence of at least three different VP7 gene clusters among rotavirus G9 strains. One
cluster was formed by the three Brazilian G9 strains, the
American strain US1205, and the strain Mc345 from Thailand; the second
cluster represented by the American strain WI61 and the Japanese strain F45; and the third cluster was formed by the Indian strain 116E (Fig.
1). Interestingly, the existence of minor
VP7 antigenic differences has recently been reported among selected G9
viruses belonging to each of the three VP7 gene clusters reported here (3, 16).
A large diversity was observed with regard to the VP4 specificity of
the Brazilian G9 strains. Although the majority of the G9 isolates
carried P[8] specificity, strains bearing P[4] or P[6]
specificity were also detected (Table 1). Thus far, rotavirus serotype
G9 has been associated with VP4 genotype P[6], P[8], P[11], or
P[19] (2, 6, 23, 25, 28). Here we found a G9 rotavirus
isolate bearing the P[4] genotype. It is believed to be the first
detection of such a G:P genotype combination. Rotavirus strains bearing
G9 specificity have been previously described in Brazil on only three
occasions: one isolate was detected in an infant (18), and
the other isolates were detected in pigs (26, 30). The
detection of rotavirus genotype G9 strains with such remarkable
frequency among diarrheal patients (15.9%) in the last 2 years
demonstrates that such a rotavirus genotype is an emerging pathogen in
this country.
Sixteen of 157 (10.2%) samples were typed as rotavirus genotype
P[9]: seven single infections and nine mixed infections (Table 3), all from outpatients. Twelve strains
were detected in 1997, eleven of them being detected during the period
from April to July and one being detected in October. Five of 12 samples were from one center in Niterói, and seven were from a
center in a wealthy area in the city of Rio de Janeiro. Three samples
were detected between July and September of 1998 in one center in Rio de Janeiro located in a middle-class area; one sample was detected in
January 1999 in a center in Rio de Janeiro that attends the poor
population of a slum.
The 16 P[9] samples were adapted to growth in primary African green
monkey kidney cells (BioWhittaker, Walkersville, Md.) and
double-stranded RNAs of such viruses were analyzed by PAGE. Interestingly, 10 of 16 viruses demonstrated a characteristic AU-1-like
electropherotype (i.e., widely spaced genes 5 and 6 and genes 10 and
11) or a mixture of an AU-1-like pattern plus an additional RNA pattern
(Fig. 2). This observation suggests a
genetic similarity between the Brazilian G3:P[9] strains and the
Japanese AU-1 virus (G3:P3[9]), which is similar to feline rotavirus
genetically (19). Since (i) 6 of 16 P[9] samples had an
AU-1-like PAGE pattern with G3:P[9] specificity and (ii) 4 of 16 P[9] samples had a mixture of an AU-1-like PAGE pattern plus an
additional PAGE pattern, it is possible that the remaining 6 P[9]
samples which did not show the AU-1-like pattern may be naturally
occurring reassortants between the AU-1-like virus and non-AU-1-like
virus.
Rotavirus P3[9] strains have been detected most commonly among
nonhospitalized children with diarrhea (7, 31).
Silberstein and colleagues (31) speculate that genotype
P[9] viruses (i) infect children as a consequence of zoonotic
infection by circulating feline rotaviruses or by reassortants formed
between feline and human group A rotaviruses such as K8 strain and thus
(ii) are attenuated to some extent in the human host and cause sporadic and mild infections in children. Unfortunately, we have no access to
the clinical data of patients who shed P[9] viruses in this study,
which might shed light on the severity of diarrhea caused by such a P
genotype. However, it is noteworthy that none of 16 patients who shed
P[9] viruses in this study required hospitalization. Before this
study, the detection of the rotavirus P[9] genotype was reported in
Brazil only once, where three isolates were detected in a collection of
fecal samples obtained between 1982 and 1994 in the state of São
Paulo (17). The detection of 11 samples in a short period
of time, 4 consecutive months within the same year, suggests the
possibility of the occurrence of one or maybe two (since the samples
came from two different cities) small outbreaks.
Of note is the finding that no G5 rotavirus strains were detected
during the 3-year study period (1997 to 1999) in two cities of the
state of Rio de Janeiro. This was particularly surprising since
the incidence of this genotype appeared to be increasing in Brazil in
the last decade (9). Is the disappearance of the genotype
G5 virus in this study area somehow related to the emergence of the
genotype G9 virus? It will be important to continue rotavirus strain
surveillance in this and other regions of Brazil to determine whether a
similar phenomenon occurs in the country. The results described here
should reinforce the importance of rotavirus G9 as an epidemiologically
important serotype and intensify the need of considering it as a
vaccine candidate.
We thank Maria Odete O. Carvalho and Giovani C. V. Costa for
supplying the stool samples used in this work; Ronald Jones, Jerri
Ross, and Mariam Wagner for their expert technical assistance; and
Albert Kapikian for encouragement throughout the study.
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