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Previous Article | Next Article 
Journal of Clinical Microbiology, April 2000, p. 1681-1683, Vol. 38, No. 4
0095-1137/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
G and P Genotyping of Rotavirus Strains Circulating
in France over a Three-Year Period: Detection of G9 and P[6]
Strains at Low Frequencies
F.
Bon,1
C.
Fromantin,1
S.
Aho,2
P.
Pothier,1,3
E.
Kohli,1,3,* and
The Azay Group
Laboratoire de
Virologie1 and Service
d'Epidémiologie et Hygiène
Hospitalière,2 Centre Hospitalier
Universitaire, and Microbiologie Médicale et Moléculaire,
Facultés de Médecine et Pharmacie,3
21034 Dijon, France
Received 23 August 1999/Returned for modification 5 November
1999/Accepted 20 December 1999
 |
ABSTRACT |
Rotavirus G and P types from 716 children with acute diarrhea in
Dijon from 1995 to 1998 and throughout France during the winter of
1997-1998 were analyzed by reverse transcription-PCR. P[8],G1
predominated, followed by P[8],G4, which emerged during the last
winter. G9 and P[6] strains were detected at low frequencies.
| |
TEXT |
Group A rotavirus is the major
etiologic agent of acute gastroenteritis in children worldwide. The two
outer capsid proteins define two different serotype specificities: G
types are determined by VP7 (gene 9), and P types are determined by VP4
(gene 4) (6). Genotyping methods are sensitive for strain
typing, notably for P typing (11), and epidemiological
studies conducted worldwide have shown that the majority of strains
were P[8],G1; P[4],G2; P[8],G3; and P[8],G4 (9).
However, other G serotypes have now been found to be common in several
regions of the world, notably, serotypes G5, G8, and G10 in Brazil
(18; V. Gouvea and N. Santos, Letter, Vaccine
17:1291-1292, 1999); G8 in Malawi (4); and G9 in
India (16) and in the United States (17). Since the role of heterotypic protection is not yet clear, strain typing is
important from the perspective of introducing polyvalent vaccines.
In France, there is no national strain surveillance system, and few
data are available concerning rotavirus type circulation. A recent
study conducted in one hospital in Paris (7) during a 1-year
survey (1997-1998) reported that 98% of 170 rotavirus isolates were G1
to G4, with type G4 predominating (60%). Here, we report strain typing
results obtained in one city (Dijon) over a 3-year period (1995 to
1998) and in 15 other cities distributed throughout France during the
1997-1998 rotavirus season. We showed that the P[8],G1 type was
predominant and that the P[8],G4 type has emerged during this period
to become very common during the winter of 1997-1998. Furthermore,
unusual strains such as G9 and P[6] could be detected at low frequencies.
Rotavirus isolates recovered from 218 children with acute
gastroenteritis in a previous study in Dijon from December 1995 to
February 1998 (3) were first analyzed. In addition, 498 stool specimens, selected randomly among 645 specimens provided by 15 hospital-based laboratories from November 1997 to May 1998, were
further typed. These laboratories, recruited for their geographic representativeness, were distributed in five regions: northeastern (three hospitals), northwestern (four), southeastern (three), southwestern-central (three), and Paris (two). To these 498 specimens were added, for analysis of the 1997-1998 rotavirus season, 55 isolates
previously typed in Dijon (northeastern region); thus, a total of 553 specimens were included. The goal of the selection was to type about
120 isolates from each region and 60 from Paris. All the samples were
from children with community-acquired gastroenteritis, and when
hospitalization was required, stool specimens were collected within
48 h to exclude nosocomial infections. Fecal specimens were
shipped to our laboratory in dry ice and were stored at 
40°C until
they were analyzed. Rotavirus RNA was extracted from 10 to 20% fecal
suspensions in phosphate-buffered saline using the QIA Amp viral RNA
kit (Qiagen, Hilden, Germany) according to the manufacturer's
protocol. G types were identified by multiplex reverse
transcription-PCR (RT-PCR) assay using primers Beg9 and End9 and,
for typing, RVG9 and the G1(aBT1), G2(aCT2), G3(aET3), and
G4(aDT4) primers (10). Specimens that could not be typed were further assayed with primers G8(aAT8) and G9(aFT9) (10) or G9(9T-9B) (5). P types were identified by multiplex
RT-PCR assay using primers con3 and con2 and, for typing, con3 and the P[8](1-T1), P[4](2-T1), and P[6](3-T1) primers (8).
The amplified products were detected by electrophoresis on a 2%
agarose gel containing 0.5 µg of ethidium bromide per ml. For
specimens not amplified by RT-PCR, a confirmatory enzyme immunoassay
(EIA) was done as previously described (15). RNAs extracted
from some specimens were resolved on a 10% polyacrylamide
discontinuous gel and stained with ethidium bromide. Nucleotide
sequencing of gene 9, for some PCR products, was carried out with the
ABI Prism BigDye Terminator Cycle Sequencing Ready Reaction kit on an
automated sequencer (model 373A; Perkin-Elmer). Statistical analysis
was performed with EPI-INFO version 6.02 software (Centers for Disease Control and Prevention, Atlanta, Ga., and World Health Organization, Geneva, Switzerland, 1994). Distributions of rotavirus types by geographic area were compared by chi-square test. All tests were two-tailed, and P values of <0.05 were considered significant.
The mean and median ages of the 218 children with acute gastroenteritis
in Dijon from December 1995 to February 1998 were 10.2 and 7.5 months,
respectively (range, 0.2 to 107.7 months; standard deviation, 11.4).
Strain P[8],G1 was predominant (169 of 218 [77.5%]). Among the
P[8],G1 isolates, three samples which could not be G typed by RT-PCR
were found to be G1 type by gene 9 sequencing (unpublished data). The
next most common strain was P[8],G4 (30 of 218 [13.8%]); strains
P[8],G3 and P[4],G2 were less common (7 and 3 of 218, respectively
[3.2 and 1.4%, respectively]). Dual infections with P[8],G1 and
P[8],G4 were found in two specimens (0.9%). Seven isolates (3.2%)
were nontypeable; all were antigen positive by EIA, but one could not
be amplified and six could not be G typed. The temporal distribution of
rotavirus types in Dijon during the 3-year period is represented in
Fig. 1. The results show the emergence of
the G4 type during the last period of the study (32.1% for the period
from December 1997 to February 1998 compared to 3.1 and 3.3% for the
same periods in the two preceding years) and the disappearance of the
P[8],G3 type since 1996.
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FIG. 1.
Temporal distribution of rotavirus types in Dijon over
the 3-year period from December 1995 to February 1998.
|
|
The mean and median ages of the 553 children with acute gastroenteritis
throughout France from November 1997 to May 1998 were 11.2 and 9 months, respectively (range, 0.1 to 135.1 months; standard deviation,
12.8). The P[8],G1 strain was predominant (309 of 553 [55.9%]),
followed by P[8],G4 (215 of 553 [38.9%]). These two strains
represented 94.8% of the isolates. Other strains were detected at low
frequencies: P[4],G2, another conventional strain (5 of 553 [0.9%]), and unusual strains (14 of 553 [2.5%]). The latter were
distributed as follows: P[4],G1 and P[8],G2 (4 and 1 of 553 [0.7
and 0.2%, respectively]), P[6],G1 and P[6],G2 (4 and 3 of 553, respectively, which gives a total of 7 P[6] strains [1.3%]); and
P[8],G9 (2 of 553 [0.4%]). The P[6] strains were detected in
three cities: Paris (three G2 strains), Limoges (three G1 strains), and
Clermont-Ferrand (one G1 strain). The two G9 strains detected in the
same city (St.-Etienne) were amplified not by the G9(FT9) primer
(10) but by the G9(9T-9B) primer (5). They had a
long electropherotype (data not shown). Nucleotide sequencing of gene 9 for these two isolates showed the same sequence and confirmed the G9
type (unpublished data). Dual infections were found in 0.4% of samples
(2 of 553). Finally, eight isolates (1.4%) that were antigen positive
by EIA could not be amplified by RT-PCR. Comparison of the distribution
of P[8],G1 and P[8],G4 strains according to age showed no
statistically significant difference. The geographic distribution of
rotavirus types (Fig. 2) showed differences between some regions: the P[8],G1 strain was less frequent in the northwestern region and more frequent in the
southwestern-central region than in the other regions; at the opposite
extreme, P[8],G4 was more frequent in the northwestern region and
less frequent in the southwestern-central region.
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FIG. 2.
Geographic distribution of rotavirus types P[8],G1;
P[8],G4; P[4],G2; P[6]; and G9 during the 1997-1998 rotavirus
season.
|
|
The results obtained here showed that P[8],G1 predominated in Dijon
from 1995 to 1998, as well as throughout France during the 1997-1998 rotavirus season. This result is consistent with numerous previous
studies (9). The next most common type was P[8],G4, and
the 3-year survey in Dijon showed clearly that this type emerged during
the winter of 1997-1998. The predominance of type G4 has been reported
in France in one hospital in Paris during the same period
(7). In our study, either P[8],G1 or P[8],G4 was
predominant according to the region. A high prevalence of the G4 type
has already been found for several other countries in Europe: the
United Kingdom (13), Finland (20), Italy
(2), and, more recently, Ireland (14), where an
increase in the prevalence of G4 isolates was observed during the
winter of 1997-1998. The two other conventional strains, P[8],G3 and
P[4],G2, were uncommon, and it is notable that P[8],G3 was not
detected after 1996. We also identified (at low frequencies) unusual
strains, among them the P[8],G9 type detected in two samples from the
same city and the P[6] type in seven samples from three different
cities, associated with G1 or G2 types. Such strains have now been
reported for children with diarrhea in several studies in
industrialized countries (1, 12, 17, 19). To our knowledge,
G9 strains have not been previously described in Europe, whereas P[6]
strains have been reported recently for the first time in Italy
(1).
Finally, in addition to the results of strain typing, this study has
allowed us to test a national strain surveillance system of
collaborating laboratories.
| |
ACKNOWLEDGMENTS |
This work was supported by grant PHRC 1997 from the Ministry of
Health and by the Programme de Recherche Fondamentale en Microbiologie et Maladies Infectieuses et Parasitaires (grant B02179, Ministry of
Education and Research).
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Microbiologie
Médicale et Moléculaire, Facultés de Médecine
et Pharmacie, 7 Bd Jeanne d'Arc, 21034 Dijon, France. Phone: 33 3 80 29 34 37. Fax: 33 3 80 29 36 04. E-mail:
Evelyne.Kohli{at}u-bourgogne.fr.
The Azay Group participants for rotavirus specimen collection are
as follows (all locations are in France): G. Agius (CHU Poitiers), E. Bingen and C. Doit (Hôpital Robert Debré, Paris), R. Colimon (CHU Rennes), F. Denis and S. Ranger-Rogez (CHU Limoges), F. Freymuth (CHU Caen), F. Eb and G. Duverlie (CHU Amiens), P. Lebon
(Hôpital St-Vincent de Paul, Paris), F. Lunel-Fabiani and S. Kouyoumdjian (CHU Angers), H. Peigue-Lafeuille (CHU Clermont-Ferrand), B. Pozetto (CHU St-Etienne), J. Puel (CHU Toulouse), M. Segondy (CHU
Montpellier), F. Stoll-Keller (Faculté de Médecine,
Strasbourg), P. Wattre (CHU Lille), and C. Zandotti (CHU Marseille).
| |
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Journal of Clinical Microbiology, April 2000, p. 1681-1683, Vol. 38, No. 4
0095-1137/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
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