Journal of Clinical Microbiology, August 1999, p. 2734-2736, Vol. 37, No. 8
0095-1137/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Detection of Porcine Rotavirus Type G9 and of a Mixture of Types
G1 and G5 Associated with Wa-Like VP4 Specificity: Evidence for
Natural Human-Porcine Genetic Reassortment
Norma
Santos,1,*
Rita
C. C.
Lima,1
Carlos M.
Nozawa,2
Rosa E.
Linhares,2 and
Vera
Gouvea1
Departamento de Virologia, Instituto de
Microbiologia, Universidade Federal do Rio de Janeiro, Rio de
Janeiro, RJ 21941-590,1 and Departamento
de Microbiologia, Universidade Estadual de Londrina, Londrina, PR
86061-970,2 Brazil
Received 1 February 1999/Accepted 19 April 1999
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ABSTRACT |
Rotavirus type G5 is a primarily porcine pathogen that has caused
frequent and widespread diarrhea in children in Brazil and in piglets
elsewhere. Initial results on the rotavirus types circulating in
diarrheic piglets in Brazil disclosed a high diversity of strains with
distinct G types including G1, G4, G5, and G9 and the novelty of
P[8], the predominant human P specificity type. Those results add
strong evidence for the emergence of new strains through natural reassortment between rotaviruses of human and porcine origins.
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TEXT |
Rotavirus type G5 strains are
primarily porcine pathogens that have occasionally been recovered from
horses, usually in combination with the P9[7] VP4 specificity type
(16, 21). Nevertheless, recent studies have demonstrated
that they are common human pathogens in Brazil, generally in
combination with the P1A[8] specificity type (9, 13).
Those findings have suggested that the human strains might have arisen
by natural reassortment among rotavirus strains of human and animal
origins (8). However, since characterization of rotaviruses
isolated from piglets or horses has not been reported in Brazil yet, it
is difficult to speculate over this matter. Knowledge of the
distribution of the G and P types circulating in the porcine population
of Brazil might bring new insights to this hypothesis. To address this
issue, we analyzed rotavirus isolates recovered from piglets in the
state of Paraná, Brazil, and verified whether G5 rotavirus
strains circulate among this species in the country and, therefore,
provide an additional clue to the occurrence of natural reassortment
between human and porcine strains.
Stool samples were collected from seven different pig farms located in
four counties in southwest Paraná, Brazil. The specimens were
obtained from piglets 21 to 35 days-old with diarrhea between March
1991 and March 1992. The piglets were raised in confinement without any
contact with animals of other species. Virus double-stranded RNA was
extracted from stool specimens and examined by polyacrylamide gel
electrophoresis (PAGE) followed by standard silver staining. The
electropherotypes of the rotavirus-positive specimens were compared to
the long and short electrophoretic patterns of group A human strains.
PCR amplifications for the identification of the G and P types of the
isolates were performed in two steps as described originally, with some
modifications (6, 10, 14, 15). Six stool specimens were
positive for rotavirus RNA by PAGE (Table
1). Four samples exhibited RNA profiles
consistent with group A rotavirus (Fig.
1). Two samples, S2 and S8, exhibited atypical profiles and were further analyzed by PCR for group B and C
rotavirus by previously described techniques (7).
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FIG. 1.
PAGE of eight porcine fecal specimens obtained in
Paraná, Brazil. L1 and L2 are reference group A human rotavirus
strains with short and long electropherotypes, respectively.
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Despite the limited number of samples analyzed, the PCR results
revealed a high diversity of G types (Table 1). The isolates presented
types G4 (two), G9 (two), and G1 (one); the sixth specimen presented a
mixture of types G1 and G5. The VP4 specificity was less diverse, with
five of the six isolates presenting one of the most common porcine
types, P[6, Gott] (Table 1) (16, 21, 24). We have added
the designation "Gott" for Gottfried-like, because our PCR
methodology clearly differentiates the P[6] genotype into two
subtypes, P[6, M37] and P[6, Gott], which correlate with the
serotypes P2A (M37-like) and P2B (Gottfried-like), respectively (14, 16). The main purpose of PCR typing is to predict the antigenic specificity of the strains. Consequently, specification of
the subtype is relevant and should be accommodated in the current genotype nomenclature until a definitive classification is adopted.
Three isolates displayed unconventional combinations of G and P types,
showing the common porcine P[6, Gott] specificity type associated
with types G1 and G9, which are G types usually described for human
isolates, whereas two isolates presented the typical porcine P[6,
Gott] G4 types. It was interesting to note that one of those typical
isolates, S2, presented an atypical group A electropherotype that
resembled the profile observed for group C rotaviruses (Fig. 1).
However, lack of recognition by group C-specific generic primers indicated that the RNA profile indeed represented the group A rotavirus
typed, either as a strain with rearranged segment 7 or, most likely, as
a mixture of two G4 P[6, Gott] strains.
Type G9 rotavirus was identified in specimens S4 and S5 collected in
July of 1991 from two piglets on farm C (Table 1). They presented the
same P specificity type and identical electropherotypes and probably
represent a single strain that circulated in the winter of 1991 on that
farm. The G9 specificity has been associated with infections usually in
humans and seldom in animals (16). It was initially
recognized as a new serotype in the United States, where it caused
approximately 9.2% of the cases of infant rotaviral disease studied at
the Children's Hospital of Philadelphia, Philadelphia, Pa., in
1983-1984 (3). It was also found sporadically in children with diarrhea in Japan and Thailand and very frequently in India, though mostly in children with asymptomatic neonatal infections (4, 18, 23). In Brazil, rotavirus type G9 has been reported only once, from a child, during a vaccine trial conducted in the northern state of Pará (17). Yet, after a silent
decade in Philadelphia, the incidence of type G9 strains achieved an
astonishing 56% of the rotavirus-associated diarrheal cases seen at
the Children's Hospital of Philadelphia in the 1995-to-1996 season
(H. F. Clark, personal communication), attesting to the typical
and well-known periodicity of group A rotavirus serotypes (11,
20). The high population density and heterogeneity of major urban
centers should favor transmission of newly adapted human strains
(8). Thus, the explosive reappearance of type G9 rotavirus
in Philadelphia might have resulted in its rapid spread to other
geographic areas, as a few G9 strains were detected in the following
season in the Midwest (20). It is of note, however, that the
single porcine G9 virus described previously, the ISU-64 strain, had
been isolated in Iowa, in the midwestern United States, in 1988 (19, 24). A second animal rotavirus of type G9 was
identified in a lamb in Scotland (strain LRV2c) in 1995 (5).
Hence, serotype G9 has circulated among humans and animals in the
United States and elsewhere for many years, a fact that extends the
likelihood that interspecies transmissions may have occurred. Although
to date, G9 has been a minor serotype in the causation of human
diarrhea, the recent identification of rotavirus type G9 in association with human and porcine diarrhea in Brazil, in studies that analyzed only limited numbers of fecal specimens, clearly expands the geographic distribution and perhaps the significance of this rotavirus serotype in
diarrheal disease.
Rotavirus type G1 is widespread and has been the most prevalent G type,
causing diarrhea in children everywhere in the last two decades
(11, 16). Nevertheless, porcine rotavirus strains belonging
to serotype G1 have been described previously for Argentina and
Venezuela (1, 2). The current discovery of type G1 in Brazil
suggests that G1 strains might also be frequent porcine pathogens in
South America.
One specimen, S8, presented a number of interesting characteristics. In
addition to G1, it contained a G5 rotavirus, demonstrating a case of
mixed infection. It displayed an atypical electropherotype, probably
due to the mixture of two strains, although only 11 segments were
discernible by PAGE (Fig. 1). A single P[8] type that was confirmed
by sequence analysis was identified in S8, suggesting the presence of a
P[8] G1 strain and a P[8] G5 strain in the fecal specimen.
Comparison of partial gene 4 sequence of the S8 strain with the partial
sequences of several rotavirus strains revealed a low similarity
between S8 and known porcine P types: only 52.7% similarity to the OSU
strain and 61.2% similarity to the Gottfried strain. Nevertheless, the
S8 sequence was 96% similar to the Wa sequence (12). A
dendrogram based on their partial gene 4 sequences that clearly
demonstrated the closer relation to gene 4 of Wa than to the porcine
gene 4 was constructed (Fig. 2).
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FIG. 2.
Phylogenetic analysis of the partial gene 4 sequences of
porcine rotavirus strains S8, Gottfried, and OSU and the human strain
Wa. The cDNA generated in the reverse transcription-PCR amplification
of a portion of gene 4 of the isolate S8 was sequenced (12),
and a dendrogram was constructed by the Clustal method.
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