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Journal of Clinical Microbiology, July 1999, p. 2373-2375, Vol. 37, No. 7
0095-1137/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Distribution of Human Rotavirus G Types
Circulating in Paris, France, during the 1997-1998 Epidemic:
High Prevalence of Type G4
Elyanne
Gault,1
Roxane
Chikhi-Brachet,1
Sandrine
Delon,1
Nathalie
Schnepf,1
Laurence
Albiges,1
Emmanuel
Grimprel,2
Jean-Philippe
Girardet,3
Pierre
Begue,2 and
Antoine
Garbarg-Chenon1,*
Laboratoire de Virologie (EA 2391, UFR
Saint-Antoine),1 Consultation de
Pédiatrie Générale et
Urgences,2 and Service de
Gastro-Entérologie Pédiatrique,3
Hôpital Armand Trousseau, 75571 Paris Cedex 12, France
Received 19 January 1999/Returned for modification 22 March
1999/Accepted 19 April 1999
 |
ABSTRACT |
Group A human rotavirus G genotypes were determined by means of
reverse transcription-PCR in 170 stool specimens from children with
acute diarrhea admitted to a Paris children's hospital during a 1-year
survey (1997 to 1998). The isolates all belonged to types G1 to G4,
with type G4 predominating (60%).
| |
TEXT |
Group A rotavirus is the main cause
of acute gastroenteritis in children worldwide. The virus possesses a
genome of 11 double-stranded RNA segments, each encoding one viral
protein. The G and P serotypes of group A rotavirus are specified by
two outer capsid proteins, respectively designated VP7 (encoded by
genome segment 7, 8, or 9, depending on the strain) and VP4 (gene 4 product). Fourteen rotavirus G serotypes have been described, and 10 have been recovered from humans (4). Epidemiological studies
based on G (VP7) serotyping or genotyping methods have indicated that
serotypes G1 to G4 are the most widespread, and that type G1 is the
most prevalent (3, 6, 7, 10, 13, 24, 27). Serotypes G1 to 4 are targeted by a rhesus rotavirus (RRV) tetravalent vaccine recently
licensed by the U.S. Food and Drug Administration (11, 15).
The vaccine provides moderate protection (~50%) against rotavirus
gastroenteritis of all severities and good to excellent protection
(~80 to 100%) against severe disease (2, 9, 17, 20, 25,
26). The perspective of using such a vaccine in Europe is
attractive, but a precise knowledge of circulating G serotypes is
crucial before and after vaccine introduction. Unusual G serotypes can
be common in some parts of the world, especially developing countries,
e.g., G9 in India (18) and G5 and G10 in Brazil (12,
21). Moreover, a recent study showed that G9 was the third most
prevalent type in the United States, with an unusually high detection
rate of 7.2% (19). To our knowledge, no data on G types
circulating in France are available. We therefore determined the
frequency and temporal distribution of human rotavirus (HRV) G types
among children admitted to a Paris children's hospital during a 1-year survey. Type G4 predominated during this period.
Between September 1997 and August 1998, 356 fecal samples from children
under 5 years of age with acute diarrhea admitted to Trousseau
Pediatric Hospital, Paris, France, were found positive for rotavirus
infection by enzyme immunoassay (Abbott Diagnostic, Rungis, France) or
electron microscopy. As usually reported in industrialized countries
(4), a seasonal pattern of infection was observed, with the
epidemic peak occurring in December (Fig. 1). At least 40% of each month's
positive samples were selected for further G-type characterization, in
order to obtain a selection representative of the epidemic
distribution. A total of 170 rotavirus-positive samples were selected;
138 were obtained from children hospitalized for community-acquired
severe acute diarrhea (mean age, 10.6 months; range, 0.1 to 48 months),
and 32 were from children who developed hospital-acquired diarrheal
illness more than 3 days after admission (mean age, 6.3 months; range,
0.3 to 52 months).
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FIG. 1.
Monthly distribution of rotavirus G types from September
1997 to August 1998. The solid line indicates the total number of
rotavirus-positive samples. The G types of at least 40% of the
rotavirus-positive samples obtained each month were determined.
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|
G types were identified by the reverse transcription-PCR assay (RT-PCR)
described by Gouvea et al. (8) with a few modifications. Stool suspensions (~10% [wt/vol] in 9
NaCl) were clarified by low-speed centrifugation, and viral RNA was extracted from 200 µl of
supernatant by using the QIAamp blood kit (Qiagen, Courtaboeuf, France)
according to the manufacturer's recommendations. Five microliters of
the RNA extract was reverse-transcribed into gene 9 (VP7) full-length
cDNA with the generic primers Beg9 and End9 (8) in a 50-µl
reaction mixture containing 20 µM EDTA, 10 mM dithiothreitol, a 0.5 mM concentration of each deoxynucleoside triphosphate, a 0.1 µM
concentration of each primer, 10 U of RNase inhibitor (Life
Technologies, Cergy, France), 200 U of SuperScript II (Life
Technologies), and 1× SuperScript buffer. After 45 min of incubation
at 45°C the reaction was stopped by adding 1 µl of 0.5 M EDTA and
150 µl of water. Five microliters of cDNA was amplified with a
mixture of G1 to G4 type-specific sense primers aBT1, aCT2, aET3, and
aDT4 (8) and a generic antisense primer, EndA, whose
sequence is conserved among G types (nucleotides 922 to 944:
5'-ATAGTATAAAATACTTGCCACCA-3'). The 50-µl reaction mixture consisted of 10 mM Tris-HCl (pH 8.3), 50 mM KCl, 2 mM
MgCl2, a 0.2 mM concentration of each deoxynucleoside
triphosphate, a 0.25 µM concentration of each primer, and 1.25 U of
AmpliTaq DNA polymerase (Perkin-Elmer, Villebon, France). Amplification
was performed in a Perkin-Elmer thermocycler (model 9700), under PCR
conditions of 35 cycles at 94°C for 30 s, 50°C for 1 min, and
72°C for 30 s. PCR products were analyzed by electrophoresis on
1.5% agarose gels. In this system, the sizes of the type-specific PCR
products were 630 bp (G1), 533 bp (G2), 255 bp (G3), and 464 bp (G4).
Rotaviruses of known G serotypes (strains Wa [G1], DS-1 [G2], SA11
and RRV [G3], and MtB2 [G4]) were used as controls in each
experiment. Negative controls consisted of rotavirus-negative stool
samples. By using this rapid extraction method and a one-step PCR
assay, 128 samples (75%) could be G typed. All but three of the
negative specimens were rescued after further RNA extraction with
RNA-PLUS (Bioprobe Systems, Montreuil, France) followed by RT-PCR
either under the same conditions or in a two-step technique. In the
latter case, gene 9 cDNA was first amplified for 25 cycles (94°C for 30 s, 55°C for 1 min, and 72°C for 30 s) with the generic
primers EndA and BegA (nucleotides 50 to 71:
5'-TGTATGGTATTGAATATACCAC-3') complementary to conserved
sequences located respectively in the 3' and 5' regions of gene 9. The
PCR product (2 µl) was then amplified with type-specific primers as
described above.
The G type was determined for 167 of the 170 rotavirus-positive
specimens (98%), all of which belonged to conventional G types 1 to 4 (Table 1 and Fig. 1). Surprisingly,
rotavirus type G4 was the most prevalent during the survey
(n = 102; 60%). Type G4 was highly predominant during
and after January 1998, when it accounted for 66 of the 90 typed cases
(73%). Type G1 was detected at a lower frequency (n = 49;
29%) and was mainly found at the beginning of the epidemic, when
it accounted for 43% of cases. Type G2 was infrequent (n = 11; 6.5%) and was only isolated at the beginning and end of the
epidemic. G2 strains had short electropherotypes when analyzed by
polyacrylamide gel electrophoresis (PAGE) (data not shown). Type G3 was
detected sporadically, in only three of the specimens (1.8%). Two
mixed G-type infections were detected (1.2%), and both were dual
infections with G1 plus G4. These mixed infections occurred during the
epidemic peak in December, when the prevalence of G1 strains was
similar to that of G4 strains. The G-type distribution was not age
dependent and was not associated with the hospital- or
community-acquired nature of the infection (Table 1). This indicated
that nosocomial rotavirus infection was probably due to continuous
introduction of community strains, as previously observed (5,
23). Only 3 of the 170 rotavirus-positive samples could not be
typed with the G1 to G4 primers. These samples were negative when
analyzed by PAGE and were also negative when tested with the G8 (aAT8)
and G9 (aFT9) type-specific primers described by Gouvea et al.
(8). In addition, the generic primers repeatedly failed to
amplify the full-length gene 9 in any of these samples. We considered
that these strains were untypeable (because of RT-PCR failure due to a
low amount of viral RNA or to the presence of strong inhibitors) rather
than belonging to unconventional G types. Thus, no rotavirus strains of
types other than G1 to G4 were identified in this survey, and their
frequency could not have exceeded 1.8%.
This 1-year survey in a Paris children's hospital provides the first
data on G types circulating in France shortly before the proposed
introduction of a rotavirus vaccine. We showed that all the rotavirus
strains analyzed belonged to the most common types, G1 to G4, a
situation encountered in the majority of industrialized countries
(3, 6, 13, 24, 27), including European states (7,
16). We only detected three type G3 strains, in keeping with
previous reports that type G3 infection is relatively uncommon in
Europe (1, 7, 14, 16); alternatively, type G3 strains may
produce a less severe illness, as our survey focused on children who
were sufficiently ill to be admitted to a hospital. One interesting finding was the high prevalence of rotavirus type G4. Type G1 is
usually the predominant strain worldwide (4, 6), and few
epidemics associated with a predominant G4 strain have been recorded
(1, 3, 14, 16). Whether or not the predominance of type G4
in our survey was restricted to the Paris area or to the 1997 to 1998 epidemic remains to be established. Additional long-term surveys
conducted as part of a strain surveillance system will be needed to
monitor the strains circulating in France, as data from a particular
site or epidemic cannot be used as an indicator of what is happening
throughout the country. In addition, such epidemics associated with a
predominant G4 strain raise concerns over vaccine efficacy; indeed, the
candidate rotavirus vaccine, RRV tetravalent vaccine, was mainly
evaluated in trials in which serotype G1 (2, 9, 17, 20) or
G3 (22) predominated.
| |
ACKNOWLEDGMENTS |
This work was supported in part by Assistance
Publique-Hôpitaux de Paris grant CRC 97135 and by the MESRT grant
Programme de Recherches Fondamentales en Microbiologie, Maladies
infectieuses et Parasitologie.
We thank J. Cohen and E. Kohli for providing the rotavirus control strains.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Laboratoire de
Virologie, Hôpital Armand Trousseau, 26 Avenue du Dr. Arnold
Netter, 75571 Paris Cedex 12, France. Phone: 33 1 44 73 62 81. Fax: 33 1 44 73 62 88. E-mail: a.chenon{at}trs.ap-hop-paris.fr.
| |
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Journal of Clinical Microbiology, July 1999, p. 2373-2375, Vol. 37, No. 7
0095-1137/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
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