Study on Aichi virus, parechovirus, and bocavirus detected in children with acute gastroenteritis in Japan, Bangladesh, Thailand, Vietnam, and Sri Lanka
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Acute gastroenteritis is one of the most common diseases in infants and children. It continues to be a significant cause of morbidity and mortality worldwide. Rotavirus, norovirus, sapovirus, adenovirus, and astrovirus have been established as the most important etiologic agents. However, the etiologic agents of more than half of acute gastroenteritis patients are undiagnosed. Recently, Aichi virus, human parechovirus (HPeV), and human bocavirus (HBoV) have also been considered to be agents of this illness. Thus, the aim of this study was to perform the molecular epidemiology of these viruses in children with acute gastroenteritis in Japan, Bangladesh, Thailand, Vietnam, and Sri Lanka. The study was carried on 1,603 fecal specimens which had been shown to be negative for rotavirus, norovirus, sapovirus, adenovirus, and astrovirus (except for 362 Sri Lankan samples which had not been screened for the 5 viruses yet) by reverse transcription - polymerase chain reaction (RT-PCR) and which were collected from infants and children with acute gastroenteritis. For study on Aichi virus, 912 samples were collected in Japan, Bangladesh, Thailand, and Vietnam during 2002-2005. For HPeV and HBoV, the study was done on 691 samples from Japan, Thailand, and Sri Lanka during 2005-2008. Screening of Aichi virus was done by a nested PCR using primer sets for amplifying the 3CD junction region. HPeV was detected by RT-PCR using a primer pair to amplify 5’UTR region of its genome and was genotyped by sequencing of the VP1 region. HBoV was screened by RT-PCR using a primer pair to amplify NP1 region of its genome and was genotyped by sequencing of the VP1/VP2 region. Of the 912 samples tested, 28 (3.1%) were positive for Aichi virus. Of these, 14 (6.5%) samples were collected from Japan, 10 (2.5%) from Bangladesh, 3 (1.6%) from Vietnam, and 1 (0.9%) from Thailand. Aichi virus strains detected from Japan, Thailand, and Vietnam belonged to genotype A, while the majority of Bangladeshi strains belonged to genotype B. Of the 28 positive samples, twelve were subjected to amplification and sequence analysis of the capsid gene of Aichi viruses. The phylogenetic tree constructed from nucleotide sequences of the capsid gene of the strains studied and reference strains demonstrated that Aichi virus strains clustered into two branches. A classification of Aichi viruses based on the capsid gene was proposed, in which lineage I consists of the Aichi virus strains detected from Japan, Thailand, Vietnam, and Germany, and lineage II includes Bangladeshi strains and a Brazilian strain. Alignment of nucleotide sequences of the capsid gene of the 17 Aichi virus strains studied and reference was done, then, 5 new primers were developed based on different nucleotides between two genotypes A and B. The newly developed primers were used in a nested PCR to examine 17 known-genotype Aichi virus strains. A nested polymerase chain reaction method using genotype-specific primers based on the capsid gene was developed to differentiate between genotypes A and B of Aichi viruses. The results showed that the PCR using newly designed genotype-specific primers could generate appropriate PCR products from all 17 samples tested, the newly developed primers could differentiate genotype A from genotype B, and all matched those obtained by nucleotide sequencing of the capsid regions. The nested PCR method using genotype-specific primers is useful and can be used for genotyping of Aichi viruses. HPeV was detected in 8.1% (20/247), 14.6% (12/82), and 8.3% (30/362) of samples collected in Japan, Thailand, and Sri Lanka, respectively. Genotyping of the detected HPeV strains showed that in Japan eighteen of the 20 strains positive for HPeV were sequenced successfully for the VP1 gene and the majority of the HPeV strains (n=15) were identified as HPeV1, and the remaining 2 as HPeV3. For the detected Thai HPeV, the capsid VP1 gene of nine strains was successfully sequenced. The studied HPeV strains clustered into 4 different genotypes from HPeV1-4, and the majority of the strains studied (5 strains) belonged to HPeV1. In addition, a rare HPeV2 strain was also detected. In Sri Lanka, the capsid VP1 gene of twenty-seven strains was successfully sequenced. The studied HPeV strains clustered into HPeV1 (11), HPeV3 (1), HPeV4 (5), HPeV5 (3). Five were determined as HPeV10, and the 2 remaining strains were identified as HPeV11. This is the first report of the circulation of HPeV in infants and children with acute gastroenteritis in Sri Lanka and the first report of HPeV10 and HPeV11. In addition, the diversity of Sri Lankan HPeV was also noted. HBoV was found in 1.6% (4/247), and 1.2% (1/82) of samples collected in Japan and Thailand, respectively. Three Japanese strains were clustered into group 1, while the remaining Japanese strain and a unique Thai strain belonged to group 3. At present, Aichi virus, HPeV, EVs and HBoV were detected by RT-monoplex PCR which use a single set of primers and can detect one target virus only. In contrast to RT-monoplex PCR, RT-multiplex PCR with different pairs of specific primers for amplifying different viral genomes in one reaction tube enables us to detect for two or more targets in a single test. In addition, RT-multiplex PCR assay is a simple and potential for rapid and cost-effective laboratory diagnosis. This study developed the RT-multiplex PCR method for detection of Aichi virus, HPeV, EVs, and HBoV in fecal specimens collected from infant and children with acute gastroenteritis. In conclusion, this is the first finding of Aichi virus in fecal specimens from Bangladesh, Thailand, and Vietnam. This is also the first finding of HPeV from children with acute gastroenteritis in Thailand and Sri Lanka. In addition, from this study, a detection of novel HPeV10 and -11 strains were firstly reported. This is the first report of detecting these kinds of viruses in fecal samples from infants and children with acute gastroenteritis by RT-multiplex PCR. The present study provides better understanding on molecular epidemiology of some less explored viral pathogens, Aichi virus, HPeV, and HBoV, detected from children with acute gastroenteritis in Asia.
発行年
2011-03-24
日本十進分類法
490
学位名
博士(保健学)
学位
doctoral
学位分野
Health Science (保健学)
学位授与機関
University of Tokyo (東京大学)
研究科・専攻
Department of International Health, Graduate School of Medicine (医学系研究科国際保健学専攻)
学位授与年月日
2011-03-24
学位授与番号
甲第27140号
学位記番号
博医第3750号
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Study on Aichi virus, parechovirus, and bocavirus detected in children with acute gastroenteritis in Japan, Bangladesh, Thailand, Vietnam, and Sri Lanka
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