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Journal of Clinical Microbiology, October 2000, p. 3876-3878, Vol. 38, No. 10
Laboratorio de Rotavirus, Instituto Nacional
de Diagnóstico y Referencia Epidemiológicos (INDRE),
Secretaría de Salud, Mexico City, Mexico
Received 1 December 1999/Returned for modification 2 May
2000/Accepted 12 July 2000
Dual typing (VP4 and VP7) of rotavirus obtained from 257 Mexican
children during three epidemiological seasons was performed by reverse
transcription-PCR. The P1G1 genotype was the most prevalent (40%),
followed by P1G3 (19%) and P2G2 (16%). Thirty-one specimens (12%)
presented mixed infections, while some genotypes were not found. This
is the first dual typing of isolates from diarrhea cases in Mexico.
Rotavirus is a worldwide cause of
severe diarrhea in young children. Genetic or antigenic differences in
VP4 (P) and VP7 (G) capsid proteins have been detected in viral
isolates (11). Since VP4 and VP7 genes segregate
independently, a binary system is used to classify rotavirus
(12). There are certain genotypes that circulate with higher
frequencies: P1 to P4 for VP4 and G1 to G4 for VP7 (5, 10, 15,
21). The combinations P1G1, P1G3, P2G2, and P1G4 are the most
frequent in Brazil, Israel, Japan, South Africa, and the United States
(16, 17, 18, 20). However, relative rates of these
combinations are different in countries like India and Egypt, where
unusual combinations of P and G alleles are also present (2, 8,
14). Rotavirus strains with different combinations may occur
simultaneously during seasonal outbreaks, giving rise to the
possibility of mixed infections and genetic rearrangements (3, 6,
9, 22).
In this study we performed dual typing of rotavirus strains gathered
from eight laboratories included in the Mexican National Network for
Rotavirus Diagnosis. The study encompassed three consecutive rotavirus
epidemic seasons from July 1994 to June 1997 in several states of
Mexico. Stool specimens from 257 children less than 5 years old were
received by the reference center (National Institute of Diagnostics and
Epidemiological Reference). VP4 (P1 to P4) and VP7 (G1 to G4)
genotyping was carried out independently by heminested reverse
transcription-PCRs as previously described (4, 7).
From all 257 samples a 1,062-bp product of the VP7 gene was observed,
which corresponds to rotavirus group A (Fig.
1). For VP4, the expected 876-bp amplicon
was obtained (not shown). The second amplification products of both
genes can be seen in Fig. 1 and 2, in
which some examples of mixed infections are also shown. One hundred two
samples (39.7%) were of the P1G1 genotype, 49 (19.0%) were P1G3, and
42 (16.3%) were P2G2. Genotypes P4 and G4 were not found in this
study. The distribution and the rates of these combinations are
summarized in Table 1.
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Copyright © 2000, American Society for Microbiology. All rights reserved.
VP4 and VP7 Genotyping by Reverse Transcription-PCR
of Human Rotavirus in Mexican Children with Acute Diarrhea
ABSTRACT
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FIG. 1.
Amplification products of the VP7 gene. Lanes 1 and 2, first amplification products (1,062 bp); lanes 3 through 5, genotype G1
(749 bp); lanes 6 and 7, genotype G2 (652 bp); lanes 8 through 10, genotype G3 (347 bp); lanes 11 and 12, mixed infections with the G1G3
and G1G2 genotypes, respectively. 
X174 digested with
HaeIII (Roche Molecular Biochemicals) was used as the
molecular size (M) standard.
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FIG. 2.
VP4 genotype amplicons. Lanes 2 and 3, dual infection
with genotype P1P2; lanes 4 and 5, genotype P2 (483 bp); lanes 6 and 7, genotype P1 (345 bp). Marker VIII (Roche Molecular Biochemicals) was
used as the molecular size (M) standard.
TABLE 1.
Distribution of VP4 and VP7 genotypes of human
rotaviruses in different States of Mexico
P1G1 was the most widely distributed combination. It was found in southeastern Mexico (Quintana Roo and Yucatan states), on the western coast (Colima and Michoacan), in the central region (Mexico City and Puebla), on the eastern coast (Veracruz), and in northern Mexico (in the Mexico-U.S. border state of Nuevo Leon). The other most frequent genotypes were P1G3 and P2G2, which were found in all studied states except Yucatan and Quintana Roo. The high frequency of P1G1 in Mexico was similar to that found in other countries, like the United States, Japan, South Africa, and Israel, and was different from that reported in India and Brazil (15, 16, 17, 21). The frequency observed for P1G3 was similar to that found in the United States, whereas the high frequency observed for P2G2 has been found solely in South Africa (17).
In Mexico, rotavirus infection peaks occurred in winter (November to
March) like in other countries (11). Genotype P1G1 occurred
during the three seasonal peaks, predominating during the last two, in
contrast with P1G3, which predominated during the first peak and
decreased in the last two. In this regard, it is worth mentioning that
Padilla-Noriega et al. (13) found genotype G3 in high
prevalence in Yucatan during the 1994-1995 outbreak, but it was absent
in the next peak (1995-1996). Genotype P2G2 was not found in the first
period, but an increasing prevalence was observed beginning with the
second period (Fig. 3). The seasonal shifts in genotype frequencies might be due to different factors, such
as genetic variation of rotavirus strains, host range modifications, host immunity, and climate changes (1, 6, 9, 11, 22).
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The genetic variation may also be a consequence of simultaneous infection of a single host with different rotavirus strains (3, 6, 19, 22). In this work, 31 samples showed more than one P or G allele, meaning that more than one rotavirus strain was present. The most frequent mixed infection was genotype P1G1G3, which suggests the simultaneous presence of P1G1 and P1G3 rotavirus strains. This result correlates with the high prevalence of those genotypes (Table 1). Mixed infections have also been reported in Brazil (30%), India (6%), the United States (3%), South Africa (3%), and Japan (1%). In this study, Mexico had the second highest percentage (12%) of dual infections reported so far, increasing the likelihood of genetic reassortment, which may yield new gene combinations (2, 3, 9, 15, 20).
Genotypes of rotavirus strains with unusual combinations of P and G
alleles were found in lower frequencies as follows: P1G2, 1.9%; P2G1,
3.1%; and P2G3, 1.5% (Table 2).
Genotype P2G1 occurred in Yucatan, Colima, Nuevo Leon, and Puebla,
while P1G2 was restricted to Colima and Yucatan and P2G3 was present
only in Michoacan, Nuevo Leon, and Yucatan. These combinations could
have been generated from genetic rearrangements of the most frequent
rotavirus genotypes, since they circulated simultaneously in those
regions. It is worth mentioning that genotype P2G1 has also been
reported at low frequencies (1%) in Brazil and Japan, while P1G2 and
P2G3 have not been reported in other countries (8, 10, 19, 20,
21).
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Partially untyped samples were also found in this study. In three samples (1.2%), two P1 and one P2 G allele could not be typed, while the P allele was not identified in 13 samples (5%) with the following G alleles: nine G1, one G3, one G1G2 (mixed infection), and two G1G3. This suggests that P alleles different from P1 to P4 segregated with G1 and G3. Thus, there is a larger heterogeneity of P alleles than of G alleles in Mexican field strains. This was also found with monoclonal antibody-based typing (13) and could be due to a higher selective pressure exerted over VP4, since it is the most external viral protein (9, 11). G alleles seemed to be more conserved, because mainly G1, G2, and G3 were found (10, 14, 15, 16, 17, 20, 21). These data support the need for including a larger set of primers to identify other P and G alleles.
In conclusion, our results confirm that there is an important variability among rotavirus strains in Mexico. This is the first report of dual typing, which allowed for identifying one of the main sources of this variability, i.e., the presence of mixed infections.
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ACKNOWLEDGMENTS |
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This work was totally supported by INDRE.
We thank Ana Flisser, Dolores Correa, and Alejandro Escobar Gutierrez for encouragement and discussion of the paper, Lizbeth Teran, Angel Barney, Juan Rojas, and Misael Mondragón for technical assistance, and M. del Pilar Bada (Veracruz), Teresa Escobar (Quintana Roo), Sandra Suarez (Michoacan), Socorro Sambrano (Monterrey), Zita Gutierrez (Puebla), Mario Gaytan (Colima), Genny Mendez and Salha (Yucatan), and Felipe Mota and Maricela Echeverria (Mexico City) for collecting and sending samples.
Araceli Rodríguez Castillo and Andrés Velasco Villa contributed equally to this work.
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FOOTNOTES |
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* Corresponding author. Mailing address: Laboratorio de Rotavirus, INDRE-SSA, Carpio 470, Colonia Santo Tomas, C.P. 11340, Mexico. Phone: 525-3-41-49-53, ext. 234. Fax: 525-3-42-06-70. E-mail: indre{at}cenids.ssa.gob.mx.
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Journal of Clinical Microbiology, October 2000, p. 3876-3878, Vol. 38, No. 10
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