Kazue Uchida

Saitama Institute of Technology, Saitama, Saitama, Japan

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Publications (13)26.39 Total impact

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    ABSTRACT: In this study, the amorphous calcium phosphate (ACP) method developed previously for calicivirus concentration from water was applied for norovirus detection from food. The viral recovery from cabbage, lettuce, or ham (10g of each) was firstly examined in seeding experiments with feline caliciviruses (FCVs). The viruses were concentrated by viral adsorption to ACP particles (0.3g) in the eluent solution (40ml) from foods, collection of the particles by centrifugation, followed by dissolution of the particles with 3.3M citric acid (3ml). In ham, FCV recovery was improved by addition of ascorbic acids into the eluent solution before ACP-particle adsorption. Quantitative real-time reverse transcription-PCR (qRT-PCR) revealed that FCV recoveries were 32-33%, 50-55%, and 37-46% from cabbage, lettuce, and ham, respectively, when seeded with 10(3)-10(4) viruses, and detection limits were estimated ∼10(3) genomic copies in all 3 foods. Subsequently, the ACP-concentration method was evaluated for norovirus (NoV) detection from these 3 foods. The recoveries and detection limit of NoVs determined by qRT-PCR were 12-41% and 10(3) (genomic copies) from cabbage, 30-57% and 10(3) from lettuce, and 20-26% and 10(4) from ham, when seeded with 10(3)-10(5) viruses. This simple method may be suitable for NoV detection from these foods.
    Journal of virological methods 10/2012; · 2.13 Impact Factor
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    ABSTRACT: A novel concentration method using minute particles of amorphous calcium phosphate (ACP) was developed for the detection of caliciviruses including norovirus and sapovirus, agents of human gastroenteritis, from water. In seeding experiments with feline calicivirus (FCV), ACP particles were able to adsorb efficiently the viruses in water, and the FCV-concentrated solution was obtained by dissolution of the virus-adsorbing ACP particles with citric acid after centrifugation. By quantitative real-time RT-PCR, the recovery efficiencies from 300 ml ultrapure water seeded with 10³, 10⁴ and >10⁵ copies of FCV were 48, 68 and >100 %, respectively. A comparative study showed that in the addition of viruses at <10⁵ copies, the recovery efficiency of our method was significantly higher (P<0.05) than that of the similar calcium flocculation-citrate dissolution method. Using our newly developed method, we successfully detected 2.1 x 10⁴ copies l⁻¹ of norovirus (each of genogroups I and II) and 5.4 x 10³ copies l⁻¹ of sapovirus (genogroups I, II, IV and V) from river water. The data suggest that our new viral concentration is a rapid, simple, cost efficient and high virus recovery method, and it can be used for routine monitoring of norovirus and sapovirus in water, especially environmental water.
    Journal of Medical Microbiology 02/2011; 60(Pt 6):780-6. · 2.30 Impact Factor
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    ABSTRACT: Human norovirus (NoV) strains cause a considerable number of outbreaks of gastroenteritis worldwide. Based on their capsid gene (VP1) sequence, human NoV strains can be grouped into two genogroups (GI and GII) and at least 14 GI and 17 GII genotypes (GI/1-14 and GII/1-17). Human NoV strains cannot be propagated in cell-culture systems, but expression of recombinant VP1 in insect cells results in the formation of virus-like particles (VLPs). In order to understand NoV antigenic relationships better, cross-reactivity among 26 different NoV VLPs was analysed. Phylogenetic analyses grouped these NoV strains into six GI and 12 GII genotypes. An antibody ELISA using polyclonal antisera raised against these VLPs was used to determine cross-reactivity. Antisera reacted strongly with homologous VLPs; however, a number of novel cross-reactivities among different genotypes was observed. For example, GI/11 antiserum showed a broad-range cross-reactivity, detecting two GI and 10 GII genotypes. Likewise, GII/1, GII/10 and GII/12 antisera showed a broad-range cross-reactivity, detecting several other distinct GII genotypes. Alignment of VP1 amino acid sequences suggested that these broad-range cross-reactivities were due to conserved amino acid residues located within the shell and/or P1-1 domains. However, unusual cross-reactivities among different GII/3 antisera were found, with the results indicating that both conserved amino acid residues and VP1 secondary structures influence antigenicity.
    Journal of General Virology 05/2006; 87(Pt 4):909-19. · 3.13 Impact Factor
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    Japanese journal of infectious diseases 03/2006; 59(1):67-8. · 1.51 Impact Factor
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    ABSTRACT: A rapid, sensitive, and specific assay to detect mumps virus RNA directly from clinical specimens using a real-time PCR assay was developed. The assay was capable of detecting five copies of standard plasmid containing cDNA from the mumps virus F gene. No cross-reactions were observed with other members of Paramyxoviridae, or with viruses or bacteria known to be meningitis pathogens. Seventy-three clinical samples consisting of throat swabs collected from patients with parotitis, and cerebrospinal fluid (CSF) collected from patients with aseptic meningitis, were examined with a real-time PCR assay developed by the authors, reverse-transcription nested-PCR (RT-n-PCR), and virus isolation using cell culture. Like the RT-n-PCR assay, the real-time PCR assay could detect mumps virus RNA in approximately 70% of both throat swabs and CSF samples, while, by tissue culture, mumps virus was isolated from only approximately 20% of CSF and 50% of throat swab samples. In addition, the real-time PCR assay could be developed easily into a quantitative assay for clinical specimens containing more than 1,800 copies of mumps virus RNA/ml by using serial dilutions of the standard plasmid. The results suggest that the real-time PCR assay is useful for identification of mumps virus infections, not only in typical cases, but also in suspected cases, which show only symptoms of meningitis or encephalitis.
    Journal of Medical Virology 04/2005; 75(3):470-4. · 2.37 Impact Factor
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    ABSTRACT: Norovirus (NV) (formerly called Norwalk-like virus) is the most common cause of acute nonbacterial gastroenteritis in humans. Recently, we reported an NV genotyping scheme based on variability in the capsid N-terminal/shell (N/S) domain gene (Katayama et al., Virology 299:225-239, 2002). We found 19 genotypes, including nine of genogroup I and 10 of genogroup II. In the present study, we investigated the molecular epidemiology of NV from 66 outbreaks that occurred in Saitama Prefecture, Japan, from 1997 to 2002. We screened 416 stool specimens by a real-time reverse transcription (RT)-PCR method (Kageyama et al., J. Clin. Microbiol. 41:1548-1557, 2003) and detected 156 NV-positive specimens, from which we amplified the capsid N/S domain gene by RT-PCR and then cloned the PCR products. After sequencing these clones, we obtained 368 sequence variants (strains). By applying our classification scheme to the strains from Saitama and other published strains, we identified a total of 31 genotypes, including an additional five genotypes for genogroup I and seven for genogroup II. Of the 31 genotypes, 26 were present in the Saitama area during that time period. These results provide additional evidence for the great diversity of human NV genotypes. Specimens from all shellfish-related infections contained multiple genotypes, including several new genotypes. On the other hand, single genotypes were observed mostly in outbreaks that originated in semiclosed communities. Thus, the number of NV genotypes in each outbreak depended on the route of transmission.
    Journal of Clinical Microbiology 08/2004; 42(7):2988-95. · 4.07 Impact Factor
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    ABSTRACT: During two winter seasons between 1999 and 2001, seven strains of influenza virus were isolated from healthy pigs in Saitama Prefecture, Japan. All isolates were identified as A (H1N2) reassortant viruses. Genetic and phylogenetic analyses indicated that they had classical swine-like hemagglutinin (HA) and internal genes, and relatively early human-like neuraminidase (NA) gene. The HA and NA genes belonged to unique clusters of Japanese swine strains.
    International Congress Series 01/2004; 1263:749-753.
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    ABSTRACT: We have developed an assay for the detection of Norwalk-like viruses (NLVs) based on reverse transcription-PCR (RT-PCR) that is highly sensitive to a broad range of NLVs. We isolated virus from 71 NLV-positive stool specimens from 37 outbreaks of nonbacterial acute gastroenteritis and sequenced the open reading frame 1 (ORF1)-ORF2 junction region, the most conserved region of the NLV genome. The data were subjected to multiple-sequence alignment analysis and similarity plot analysis. We used the most conserved sequences that react with diverse NLVs to design primers and TaqMan probes for the respective genogroups of NLV, GI and GII, for use in a real-time quantitative RT-PCR assay. Our method detected NLV in 99% (80 of 81) of the stool specimens that were positive by electron microscopy, a better detection rate than with the two available RT-PCR methods. Furthermore, our new method also detected NLV in 20 of 28 stool specimens from the same NLV-related outbreaks that were negative for virus by electron microscopy. Our new assay is free from carryover DNA contamination and detects low copy numbers of NLV RNA. It can be used as a routine assay for diagnosis as well as for elucidation of the epidemiology of NLV infections.
    Journal of Clinical Microbiology 05/2003; 41(4):1548-57. · 4.07 Impact Factor
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    ABSTRACT: Mumps virus (MuV) strains isolated in Saitama Prefecture, Japan, from 1997 to 2001, were examined by analyzing the SH and the F gene nucleotide sequences. The results of the SH gene analysis showed that only genotype G was found in 2001 as well as in 2000, and that genotype J, which we proposed as a new genotype in a previous study, was from a different lineage than the genotype J described by Tecle et al. (J. Gen. Virol. 82, 2675-2680). We therefore, propose to rename the genotype as K to avoid confusion. Then, the F gene of genotypes G, H, and K strains were analyzed together with previously reported strains in this study. The results of phylogenetic analysis of the F gene nucleotide sequences showed that these strains formed a cluster as described by the SH gene analysis. Alignment of the F amino acid sequences showed that the F protein was well conserved among strains of different genotypes with a few amino acid differences. These results provide better information for the characterization of contemporary MuV strains in Japan.
    Microbiology and Immunology 02/2003; 47(2):167-72. · 1.55 Impact Factor
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    ABSTRACT: "Norwalk-like viruses" (NLV), a member of the family Caliciviridae, are the major causative agents of acute gastroenteritis and are genetically divided into two groups, genogroup I (GI) and genogroup II (GII). We have determined the complete nucleotide sequences of 10 new NLV strains. Using this information together with eight known NLV sequences, the criteria to further classify genotypes of NLV were investigated. Validation of the topological error based on the bootstrap value and the branch length (distance) allowed us to identify two potential subgenomic regions suitable for the genotyping. They were the putative 3D-like RNA-dependent RNA polymerase (polymerase) and the capsid N-terminal/Shell domains (capsid N/S domain). When the distance distribution analysis was performed, the polymerase-based classification did not separate the strains into internal clusters within the genogroup. Furthermore, a diversity plot analysis of the complete nucleotide sequences of WUG1, a NLV GI strain, and Saitama U1, a NLV GII strain, indicated that the genotype was different between the polymerase and capsid N/S domain, suggesting that these strains are the genetic recombinants. Therefore, polymerase is not suitable for genotyping. On the other hand, the clustering based on the capsid N/S domain successfully distinguished the NLV as well as the grouping based on the antigenicity, as determined by both antigen and antibody ELISAs with recombinant virus-like particles. As the nucleotide sequences of the primers for the capsid N/S domain are highly conserved among the NLV, the amplification of the unknown genotype can be easily performed. This method will facilitate global surveying as well as epidemiologic study on NLV.
    Virology 09/2002; 299(2):225-239. · 3.37 Impact Factor
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    ABSTRACT: Although various cell lines have been used for virus isolation, few study of virus isolation using MRC-5 cell, a human embryonic lung fibroblasts, have been reported in Japan. MRC-5 and other cell lines (Caco-2, Vero, RD-18s, LLC-MK2, HeLa, MDCK, FL, B95a and HMV-II), and suckling mouse were compared for isolation of viruses from clinical specimens. A total of 3,284 specimens, collected from clinics and hospitals in Saitama Prefecture between July 1997 and August 2001, were inoculated in these cells. A total of 1,252 viral strains were isolated and 1,190 viral strains of these were identified. MRC-5 detected 209 of specimens positive for various viruses. As for adenovirus, a total of 132 viral strains were isolated using cell lines described above, and 100 of 132 viral strains were isolated in MRC-5. MRC-5 showed the highest sensitivity for isolation of adenovirus 3 and 7 (79.1% and 100%) of all other cells. The sensitivity in isolation of these viruses in HeLa was 58.1% and 50.0%, respectively. It showed that MRC-5 is able to isolate enterovirus, especially coxsackie virus A16 and enterovirus 71 with a high sensitivity (85.7% and 73.7%). RD-18s detected 35.7% and 26.3% of coxsackie virus A16 and enterovirus 71 isolates, LLC-MK2 detected 60.7% and 47.4%, and Vero detected 48.6% and 52.6%, respectively. Coxsackie virus B group was not isolated, except for a few coxsackie virus B 5 strains. Enteroviruses except coxsackie virus A16 and enterovirus 71 were isolated more frequently in Caco-2 and RD-18s. Seven hundred thirteen strains of influenza viruses were isolated in MDCK and Caco-2, but none was isolated in MRC-5. It was probably due to the maintenance medium without trypsin. The isolation rate of herpes simplex virus in Vero was 88.9% and MRC-5 showed 77.8%, it was high secondary to Vero by MRC-5. However, the CPE was detected in a few days in MRC-5, it was earlier than in Vero. The MRC-5 is possible to be maintained without changing the maintenance medium and passaged for 2 weeks, and clear CPE was observed. On the other hand, the disadvantages in using the MRC-5 were that the passage was limited and that the split ratio was only 1:2. However, the MRC-5 was used successfully for virus isolation, especially coxsackie virus A16, enterovirus 71 and adenoviruses, from clinical specimens.
    Kansenshogaku zasshi. The Journal of the Japanese Association for Infectious Diseases 07/2002; 76(6):432-8.
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    ABSTRACT: Norwalk-like viruses (NLV) are a major causative agent of nonbacterial gastroenteritis. There are still many NLV strains that are refractory to gene amplification by ordinary reverse transcription-polymerase chain reaction. This is due mainly to the genetic diversity among NLV, especially mismatches in the primer sequences, which limits this technique in clinical utility. In this study, improved primer sets based on the capsid region, to detect both genogroup I and II NLV by genogroup-specific manner, were developed. When stool specimens from gastroenteritis patients, that were positive for NLV by electron microscopy, were tested by this new primer set, all specimens were positive by RT-PCR. Primers described previously for RdRp and capsid protein were capable of amplifying the specimens by 31 and 77%, respectively. Therefore, new primer sets are extremely useful for the amplification and rapid diagnosis of nonbacterial gastroenteritis due to NLV as well as for epidemiological studies.
    Journal of Virological Methods 03/2002; 100(1-2):107-14. · 1.90 Impact Factor
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    ABSTRACT: AH1-HA, AN1-NA and AH5-HA genes were not. The results suggested that the patient was infected simultaneously by both influenza A (H3N2) and B viruses. The levels of AH3-HA and B-HA genes in the original specimen were assessed by real-time quantitative PCR. Briefly, viral RNA was extracted from the specimen as described above and 50 μl of complementary DNA (cDNA) was synthesized using SuperScriptTM II reverse transcriptase (Invitrogen, Carlsbad, Calif., USA) from 20 μl of RNA solu- tion. Real-time PCR was performed at Tokyo Metropolitan Institute of Public Health as reported previously (2) with some modifications on primers and probes. The numbers of copies of influenza AH3-HA and B-HA genes in 10 μl of cDNA