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Journal of Clinical Microbiology, July 2001, p. 2722-2724, Vol. 39, No. 7
Department of Gastroenterology and Clinical
Nutrition, Murdoch Childrens Research Institute, Royal Children's
Hospital, Victoria, Australia
Received 21 December 2000/Returned for modification 30 March
2001/Accepted 7 May 2001
Reverse transcription-PCR and sequence analysis identified
calciviruses in 32 of 60 stool specimens (negative for other enteric pathogens) obtained from children admitted to our hospital with acute
gastroenteritis. The overall annual incidence rate for calcivirus was
9% (32 of 354 children). Molecular analysis identified 30 "Norwalk-like virus" genogroup II (predominantly Lordsdale cluster) and 2 "Sapporo-like virus" strains.
Human calciviruses are the most
common cause of nonbacterial gastroenteritis outbreaks in adults and
older children worldwide (2, 13) and have been estimated
to cause 95% of outbreaks of food-related viral gastroenteritis in the
United States, representing millions of gastroenteritis episodes each
year (8). Little is known about the importance of these
viruses as causes of sporadic acute gastroenteritis in young children
requiring admission to a hospital (5).
Human calciviruses can be divided into two genera: "Norwalk-like
viruses" (NLVs) and "Sapporo-like viruses" (SLVs)
(2). NLVs are a genetically diverse group of viruses that
can be classified phylogenetically into two distinct genogroups:
genogroup I, which includes Norwalk virus, and genogroup II, which
includes Lordsdale and Snow Mountain viruses. NLVs are the major cause
of outbreaks and sporadic gastroenteritis in adults (2,
13). SLVs have been associated mainly with gastroenteritis in
nonhospitalized children (9). The importance of calcivirus
infection in child health in general is still largely unknown.
The objectives of this study were to determine the minimum prevalence
of human caliciviruses in stools from young children admitted to the
Royal Children's Hospital, Melbourne, Australia, with acute
gastroenteritis between January and December 1999 and to characterize
the types and distribution of calicivirus strains identified.
A total of 354 stool specimens were collected from all children under
the age of 5 years. Routine diagnostic tests identified rotavirus
(71.5%), astrovirus (5.7%), adenovirus (3.7%), and other pathogens
(including bacterial and parasitic pathogens [2.8%]). The remaining
specimens (n = 60), where no etiologic agent was identified, were tested for the presence of calicivirus using two
reverse transcription-PCR protocols that amplify either a 319- or
321-base fragment (P289 and P290 primers) or a 213-base fragment
(degenerate primer set) of the RNA polymerase gene (2, 6).
A total of 32 of 60 samples were positive by one or both of the reverse
transcription-PCR methods. The nucleotide sequences of these products
were determined by direct cycle sequencing and analyzed using E-CLUSTAL
W (available through the Australian National Genomic Information
Service, University of Sydney).
Strains segregated genetically into three distinct NLV genogroup II
clusters (n = 30) and two clusters of the SLV genera
(n = 2). The majority of the genogroup II strains (26 of 30) belonged to the Lordsdale cluster, while three of the remaining
strains (RCH 99-8, -10, and -18) were assigned to the Snow Mountain
cluster and one was assigned to the rare Hillingdon cluster (RCH
99-27). Two strains (RCH 99-6 and -19) belonging to the SLV genera
exhibited most similarity to the Manchester and Sapporo clusters, respectively.
We conducted phylogenetic analysis of 22 strains for which a
220-nucleotide sequence was obtained using the P289-P290 primer pair
(Fig. 1). These strains clearly
segregated into three distinct NLV genogroup II clusters (Lordsdale,
Snow Mountain, and Hillingdon) and two SLV clusters (Fig. 1). The
strains not included in this analysis were those for which sequence
information was obtained solely using the degenerate primer pair
(strains RCH 99-7, -11, -16, -17, and -29) and those for which less
than the 220-nucleotide sequence was obtained using the P289-P290
primer pair (strains RCH 99-2, -4, -10, -24, and -30).
0095-1137/01/$04.00+0 DOI: 10.1128/JCM.39.7.2722-2724.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Molecular Detection of Human Calicivirus in Young
Children Hospitalized with Acute Gastroenteritis in Melbourne,
Australia, during 1999
ABSTRACT
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FIG. 1.
Phylogenetic tree of sequences of a 220-nucleotide
region of the RNA polymerase gene (open reading frame 2) of 22 calicivirus isolates collected in Melbourne during 1999 and 16 reference strains available in GenBank. Distances were calculated by
the Jukes-Cantor method, available in the MEGA analytical package
(7), and trees were drawn using the neighbor-joining
method. Reference strains were representative of clusters from both the
NLV and SLV genera. The GenBank accession numbers for the reference
strains are as follows: for Snow Mountain, NCRNAPBM1; for Camberwell,
U46500; for Lordsdale, x86557; for Hawaii, HCU07611; for Mexico,
HCU22498; for Melksham, x81879; for Desert Shield, DSU04469; for
Hillingdon, AB020558; for Southampton, L07418; for Norwalk, M87661; for
KY89, L23828; for Houston, U95644; for Manchester, x86560; for Sapporo,
HCU65427; and for London, U95645.
This study highlights the importance of calicivirus as a causative agent of sporadic cases of acute gastroenteritis in young children who require hospitalization. The overall annual incidence rate of calicivirus infection during 1999 in Melbourne children under 5 years of age was 9% (32 of 354 children). This figure represents a minimum incidence rate, since only fecal specimens negative for other enteric pathogens were examined. The occurrence of mixed infections remains unknown. Our study shows good agreement with previous surveys of the incidence of calicivirus infection in children hospitalized with acute gastroenteritis in France, China, and South Africa (1, 11, 12). In comparison, surveillance studies of young children with mild diarrhea from community or outbreak settings detected calicivirus in 19 to 88% of such cases in various countries, including Finland, Canada, and Mexico (3, 4, 9, 10).
The extent of genetic diversity in strains isolated from children admitted to our hospital was consistent with results for adults from whom over 40 distinct strains have been identified from more than 70 outbreaks in the United States and Australia (2, 13). Our findings that NLV genogroup II strains were the dominant type (identified in 94% of positive specimens) is in agreement with those of previous studies of NLV infections in pediatric populations in France, Finland, and Canada (1, 4, 9). Most genogroup II strains identified in our study (84%) belonged to the Lordsdale cluster, which has a worldwide distribution and is prominent in many countries, including Australia, the United States, and Canada (2, 4, 13). The rare Hillingdon genogroup II cluster has been identified previously only in outbreak specimens from Japan (GenBank accession number AB020558), Canada (4), and Norway (GenBank accession number x89034). Overall, the divergence in NLV strains identified in the pediatric population in Melbourne appears to be a reflection of the genetic diversity present in the broader general community.
SLV strains appear uncommon as a cause of severe gastroenteritis in young children requiring hospitalization, being detected in only two samples in this study, perhaps reflecting the belief that SLV-associated gastroenteritis is less severe than NLV-associated gastroenteritis (9, 10). SLVs were also relatively uncommon (9 of 154 specimens) as a cause of outbreaks or of sporadic gastroenteritis in adults and older children not requiring hospitalization in Melbourne from 1986 to 1996 (13).
In conclusion, our results show that human caliciviruses, predominantly genogroup II of the NLVs, are an important cause of sporadic cases of acute gastroenteritis in infants and young children requiring hospitalization in Melbourne, Australia, during January to December 1999. Further epidemiological studies are required to identify whether the predominance of NLVs of genogroup II observed in this 1-year study persists over a longer period.
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ACKNOWLEDGMENTS |
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This study was supported by a research fellowship to C.D.K. and a project grant to R.F.B. from the Murdoch Childrens Research Institute.
We thank Rebecca Fankhauser and Jacqueline Noel for assistance with calicivirus detection and providing positive control samples.
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FOOTNOTES |
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* Corresponding author. Mailing address: Department of Gastroenterology and Clinical Nutrition, Murdoch Childrens Research Institute, Royal Children's Hospital, Flemington Rd., Parkville, Victoria 3052, Australia. Phone: (613) 9345-5060. Fax: (613) 9345-6240. E-mail: Kirkwooc{at}cryptic.rch.unimelb.edu.au.
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Journal of Clinical Microbiology, July 2001, p. 2722-2724, Vol. 39, No. 7
0095-1137/01/$04.00+0 DOI: 10.1128/JCM.39.7.2722-2724.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
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