[show abstract][hide abstract] ABSTRACT: Sapovirus (SaV) is an agent of gastroenteritis for humans and swine, and is divided into five distinct genogroups (GI-GV) based on its capsid gene sequences. Typical methods of SaV detection include electron microscopy (EM), enzyme-linked immunosorbent assay (ELISA), and reverse transcription-polymerase chain reaction (RT-PCR). A novel TaqMan-based real-time RT-PCR assay was developed that is sensitive and has the ability to detect the broad range of genetically diverse human SaV strains. A nucleotide alignment of 10 full-length SaV genome sequences was subjected to similarity plot analysis, which indicated that the most conserved site was the polymerase-capsid junction in open reading frame 1 (ORF1). Based on multiple alignments of the 27 available sequences encoding this junction, we designed sets of primers and TaqMan MGB probes that detect human SaV GI, GII, GIV, and GV sequences in a single tube. The reactivity was confirmed with SaV GI, GII, GIV, and GV control plasmids, and the efficiency ranged from 2.5 x 10(7) to 2.5 x 10(1) copies per tube. Analysis using clinical stool specimens revealed that the present system was capable of detecting SaV GI, GII, GIV, and GV sequences, and no cross-reactivity was observed against other enteric viruses, including norovirus (NoV), rotavirus, astrovirus, and adenovirus. This is the first real-time RT-PCR system that could detect all genogroups of human sapoviruses.
Journal of Medical Virology 11/2006; 78(10):1347-53. · 2.37 Impact Factor
[show abstract][hide abstract] ABSTRACT: We recently determined the ORF1 cleavage map of Mc10, a human sapovirus (SaV) strain, as follows: NH2-p11-p28-p35(NTPase)-p32-p14(VPg)-p70(Pro-Pol)-p60(VP1)-COOH. This cleavage was dependent on the viral encoded 3C-like protease. To identify the cleavage site of SaV ORF1, putative p70 (Pro-Pol) and p14-p70 (VPg-Pro-Pol) were expressed as N-terminal GST and C-terminal 6 x His-tag fusion proteins in Escherichia coli, and the expressed products were analyzed by SDS-PAGE and Western blotting. Our results indicated that the efficient proteolytic cleavage occurred between p14 (VPg) and p70 (Pro-Pol), and N-terminal amino acid sequencing revealed that the cleavage site was between E(1055) and A(1056). In contrast, the p70 (Pro-Pol) was not further cleaved. We also found that SaV protease cleaved the Q/G site within the rhinovirus 3C protease recognition site. Site-directed mutagenesis in a conserved GDCG motif of the protease completely abolished these proteolytic activities. This is the first report to identify the cleavage site of the SaV ORF1 polyprotein.
Archives of Virology 01/2006; 150(12):2539-48. · 2.03 Impact Factor
[show abstract][hide abstract] ABSTRACT: The genome of Sapovirus (SaV), a causative agent of gastroenteritis in humans and swine, contains either two or three open reading frames (ORFs). Functional motifs characteristic to the 2C-like NTPase (NTPase), VPg, 3C-like protease (Pro), 3D-like RNA-dependent RNA polymerase (Pol), and capsid protein (VP1) are encoded in the ORF1 polyprotein, which is afterwards cleaved into the nonstructural and structural proteins. We recently determined the complete genome sequence of a novel human SaV strain, Mc10, which has two ORFs. To investigate the proteolytic cleavage of SaV ORF1 and the function of protease on the cleavage, both full-length and truncated forms of the ORF1 polyprotein either with or without mutation in (1171)Cys to Ala of the GDCG motif were expressed in an in vitro coupled transcription-translation system. The translation products were analyzed directly by sodium dodecyl sulfate-polyacrylamide gel electrophoresis or by immunoprecipitation with region-specific antibodies. The ORF1 polyprotein was processed into at least 10 major proteins: p11, p28, p35, p32, p14, p70, p60, p66, p46, and p120. Seven of these products were arranged in the following order: NH(2)-p11-p28-p35(NTPase)-p32-p14(VPg)-p70(Pro-Pol)-p60(VP1)-COOH. p66, p46 and p120 were precursors of p28-p35 (NTPase), p32-p14 (VPg), and p32-p14 (VPg)-p70 (Pro-Pol), respectively. Mutagenesis in the 3C-like protease motif fully abolished the proteolytic activity. The cleavage map of SaV ORF1 is similar to those of other heretofore known members of the family Caliciviridae, especially to rabbit hemorrhagic disease virus, a member of the genus Lagovirus.
Journal of Virology 07/2005; 79(12):7283-90. · 5.08 Impact Factor
[show abstract][hide abstract] ABSTRACT: Stool specimens collected between November 2002 and April 2003 from hospitalized infants with acute gastroenteritis from four distinct geographical regions in Thailand were examined for norovirus (NoV) and sapovirus (SaV) by reverse transcription-PCR and sequence analysis. Of the 80 specimens examined, we identified 11 NoV and 9 SaV single infections, and 3 NoV/SaV mixed infections. The majority of NoV strains (64%) belonged to genogroup II/ genotype 4 (GII/4; Lordsdale cluster). Other NoV strains co-circulating belonged to GII/1, GII/3, GII/6, and one new genotype cluster (GII/New). The majority of SaV strains (83%) were from the Manchester cluster. One isolated SaV strain represented a recently discovered novel genogroup within the SaV genus (SG-V), and another isolated SaV strain represented a novel SaV genogroup II cluster.
Japanese journal of infectious diseases 01/2005; 57(6):276-8. · 1.51 Impact Factor
[show abstract][hide abstract] 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
[show abstract][hide abstract] ABSTRACT: Replication of positive-strand caliciviruses is mediated by a virus-encoded RNA-dependent RNA polymerase (RdRp). To study the replication of Norovirus (NV), a member of the family Caliciviridae, we used a recombinant baculovirus system to express an enzymatically active RdRp protein from the 3D region of the NV genome and defined conditions for optimum enzymatic activity. Using an RNA template from the NV 3' genomic region, we observed similar levels of enzymatic activity in assays with and without a poly(A) tail. RdRp activity was not significantly affected by the addition of an RNA primer to the reaction mixture. Thus, the NV RdRp exhibited primer- and poly(A)-independent RNA polymerase activity. While the RdRp inhibitor phosphonoacetic acid inhibited NV RdRp activity, another gliotoxin did not. The active recombinant NV RdRp will be of benefit to studies of NV replication and will facilitate the development of specific inhibitors of NV proliferation.
Journal of Virology 05/2004; 78(8):3889-96. · 5.08 Impact Factor
[show abstract][hide abstract] 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
[show abstract][hide abstract] 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.
[show abstract][hide abstract] ABSTRACT: A rapid and efficient RT-PCR with fluorogenic probe(TaqMan-PCR) was developed for detection of Norwalk virus(NV) genomes in clinical specimens. We designed NV genogroup specific primers and fluorogenic probes in the junction of open reading frame (ORF)1 and ORF2. Eighty specimens from patients of gastroenteritis, in which NV-like particles were detected by electron microscopy, were examined by TaqMan-PCR and RT-PCR using primer sets previously reported; two sets in RdRp region of NV and one set in capsid region. TaqMan-PCR detected NV genome from 79 of 80 specimens(98.8%); this method showed the highest sensitivity of all other RT-PCR tested. Moreover, TaqMan-PCR was considered to be useful to recognize genogroup of NV correctly.
Nippon rinsho. Japanese journal of clinical medicine 07/2002; 60(6):1181-7.
[show abstract][hide abstract] 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
[show abstract][hide abstract] ABSTRACT: Genotype 2a hepatitis C virus (HCV) has different characteristics from genotype 1b, such as responsiveness to interferon therapy. Such type-specific characteristics appear to be due to differences in the HCV genome sequence. The complete sequences of genotype 2a HCV genome isolated from four patients with chronic hepatitis C were determined, and nucleotide and deduced amino acid sequences were compared within genotype 2a, as well as between genotype 2a and 1b. Whereas the amino acid sequence similarity of the core region was highest within genotype 1b, the NS3 and NS4B regions of exhibited greater similarity than the core region in genotype 2a. The serine protease and helicase motifs in the NS3 region were well conserved in genotype 2a to the same degree as in genotype 1b. However, the putative secondary structure of 2a isolates was significantly different from that of the 1b isolates. Analysis of amino acid similarity between genotypes 2a and 1b revealed the lowest degree of similarity in the E1 region, followed by the NS2 and NS5A region. Sequences of genotype 2a in the interferon-sensitivity determining region (ISDR) located in the NS5A region had a deletion of four amino acids compared with that of genotype 1b. When the ISDR of the genotype 2a was aligned for maximal similarity, it exhibited similarity of only 52.5-55.0% when compared with that of HCV-J, which belongs to genotype 1b. These findings for the entire sequences of genotype 2a isolates will contribute to virological studies of HCV.
Journal of Medical Virology 09/2001; 64(4):466-75. · 2.37 Impact Factor
[show abstract][hide abstract] ABSTRACT: Translational initiation of hepatitis C virus (HCV) genome RNA occurs via its highly structured 5' noncoding region called the internal ribosome entry site (IRES). Recent studies indicate that HCV IRES and 40 S ribosomal subunit form a stable binary complex that is believed to be important for the subsequent assembly of the 48 S initiation complex. Ribosomal protein (rp) S9 has been suggested as the prime candidate protein for binding of the HCV IRES to the 40 S subunit. RpS9 has a molecular mass of approximately 25 kDa in UV cross-linking experiments. In the present study, we examined the approximately 25-kDa proteins of the 40 S ribosome that form complexes with the HCV IRES upon UV cross-linking. Immunoprecipitation with specific antibodies against two 25-kDa 40 S proteins, rpS5 and rpS9, clearly identified rpS5 as the protein bound to the IRES. Thus, our results support rpS5 as the critical element in positioning the HCV RNA on the 40 S ribosomal subunit during translation initiation.
Journal of Biological Chemistry 07/2001; 276(24):20824-6. · 4.65 Impact Factor
[show abstract][hide abstract] ABSTRACT: The 5' noncoding region (NCR) of hepatitis C virus (HCV) contains an internal ribosome entry site for translation initiation. Cellular proteins (e.g. La, polypyrimidine tract-binding protein, and p25) that interact with HCV 5' NCR have been implicated in facilitating efficient internal initiation. The 5' NCR may also contain RNA structures and specific RNA sequences that interact with cellular proteins to promote RNA replication. UV crosslinking experiments revealed a 43-kDa cellular protein (p43) also interacts with the HCV 5' NCR. Further UV crosslinking experiments with deletion mutants of HCV 5' NCR demonstrated that p43 bound specifically to the 5'-terminal stem-loop of the HCV 5' NCR. Achromobactor proteinase I digests, competition experiments, and immunoprecipitation confirmed that p43 was identical to human poly(rC)-binding protein 2 (PCBP2). We prepared a PCBP2-immunodepleted rabbit reticulocyte lysate with an anti-PCBP2 antibody. Translation activity promoted by the HCV internal ribosome-entry site was the same in PCBP2-depleted lysates as in mock-depleted lysates. In conclusion, PCBP2 specifically interacted with the 5' terminus of HCV genome but had no effect on HCV translation. We speculate that PCBP2's interaction with HCV 5' NCR may be involved in the replication-initiation complex of HCV.
Virus Research 02/2001; 73(1):67-79. · 2.75 Impact Factor
[show abstract][hide abstract] ABSTRACT: With the aim of elucidating evolutionary features of GB virus C/hepatitis G virus (GBV-C/HGV), molecular evolutionary analyses were conducted using the entire coding region of this virus. In particular, the rate of nucleotide substitution for this virus was estimated to be less than 9.0 x 10(-6) per site per year, which was much slower than those for other RNA viruses. The phylogenetic tree reconstructed for GBV-C/HGV, by using GB virus A (GBV-A) as outgroup, indicated that there were three major clusters (the HG, GB, and Asian types) in GBV-C/HGV, and the divergence between the ancestor of GB- and Asian-type strains and that of HG-type strains first took place more than 7000-10,000 years ago. The slow evolutionary rate for GBV-C/HGV suggested that this virus cannot escape from the immune response of the host by means of producing escape mutants, implying that it may have evolved other systems for persistent infection.
Journal of Molecular Evolution 05/1999; 48(4):383-9. · 2.15 Impact Factor
[show abstract][hide abstract] ABSTRACT: Translation initiation of hepatitis C virus (HCV) RNA is controlled by an internal ribosome entry site (IRES) contained in 5' noncoding region (NCR) and in several nucleotides of the coding region. The ability of a 25-kilodalton cellular protein (p25) to bind the HCV 5' NCR is correlated with the efficiency of translation initiation of HCV RNA, indicating that this protein plays a critical role in HCV translation (S. Fukushi, C. Kurihara, N. Ishiyama, F. B. Hoshino, A. Oya, and K. Katayama, J Virol 71, 1662-1666, 1997). We have extended the study for identification of the IRES region required for p25 binding. For this purpose, we have performed UV cross-linking competition analyses using 5'- or 3'- deleted mutants of the HCV 5' NCR as competitor RNAs for binding of p25 to wild-type HCV 5' NCR. Competitor RNAs lacking nucleotides (nt) 47-74 or nt 279-331 did not inhibit p25 binding to the HCV IRES, indicating that these regions are necessary for interaction of the p25 and HCV IRES. Since p25 binding was not observed in the IRES elements of encephalomyocarditis virus and poliovirus in UV cross-linking competition analyses, the p25 binding may be specific for the HCV IRES. p25 bound to the HCV IRES was detected when a purified 40S ribosomal subunit was used for UV cross-linking experiment, indicating that p25 is one of 40S ribosomal subunit proteins. These results reveal an unique interaction between the 40S ribosomal subunit and HCV IRES to contribute to translation initiation of the HCV genome.
[show abstract][hide abstract] ABSTRACT: A new quantitative reverse transcription-polymerase chain reaction (RT-PCR) method is described for analyzing the amount of GB virus-C (GBV-C)/hepatitis G virus (HGV) RNA in serum. This multicyclic RT-PCR (MRT-PCR) method used oligonucleotide primers deduced from the 3' noncoding region (3'NCR) that is highly conserved among GBV-C/HGV isolates. Quantitation of GBV-C/HGV RNA using MRT-PCR ranged between 10(2) and 10(10) copies/ml when PCR cycle number was regulated at exponential amplification of the products. Competitive RT-PCR (CRT-PCR) was carried out with mutant RNA and sample that had been measured by MRT-PCR. Quantitation of GBV-C/HGV RNA using both methods agreed. MRT-PCR detected viral RNA in a single step PCR, and demonstrated a high degree of sensitivity that was equal to that of the RT-PCR procedure, which used nested primers deduced from the non-structural (NS) 3 region. The MRT-PCR method for quantitation of GBV-C/HGV RNA in serum may prove useful for diagnosis.
Journal of Virological Methods 11/1998; 74(2):185-91. · 1.90 Impact Factor
[show abstract][hide abstract] ABSTRACT: The genomes of nine GBV-C/HGV isolates from Japanese chronic hepatitis patients were fully sequenced and characterized. They shared 85% nucleotide sequence homology with previously characterized isolates from the US and West Africa. Homology studies and phylogenetic analyses showed that the Japanese isolates formed a third group distinct from the established groups 1 and 2. The genetic distances between the three groups of GBV-C/HGV were very similar to the distances between the two classical swine fever virus (CSFV) serotypes, which suggested that they might belong to a separate GBV-C/HGV serotype. Plot similarity analysis comparing the three groups exposed relatively conserved terminal non-coding regions. Hairpin structures predicted in the Japanese isolates are probably involved in viral replication. The region coding E1-E2-NS-2 showed the least similarity (80%); in HCV the similarity here is only 50% due to its hypervariability. NS-3 and NS-5b that respectively encode the helicase/protease and RNA-dependent RNA polymerase, had a high degree of amino acid homology, suggesting a high degree of functional constraint in this region. The NS-5b nucleotide sequence was highly conserved perhaps because of constraints from RNA secondary structure and/or an open reading frame in the negative strand.
Archives of Virology 02/1998; 143(6):1063-75. · 2.03 Impact Factor