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Naokazu Takeda

Publications and source records attributed to Naokazu Takeda.

18 recordsLinked to original sources
PubMed2003

Characterization of self-assembled virus-like particles of human polyomavirus BK generated by recombinant baculoviruses.

The major structural protein of the human polyomavirus BK (BKV), VP1, was expressed by using recombinant baculoviruses. A large amount of protein with a molecular mass of about 42 kDa was synthesized and identified by Western blotting. The protein was detected exclusively in the nuclei by immunofluorescent analysis and it was released into culture medium. The expressed BKV VP1 protein was self-assembled into virus-like particles (BK-VLPs) with two different sizes (50 and 26 nm in diameter), which migrated into four different bands in CsCl gradient with buoyant densities of 1.29, 1.30, 1.33, and 1.35 g/cm(3). The immunological studies on the BK-VLPs suggested that they have similar antigenicity with those of authentic BKV particles. Cryoelectron microscopy and 3D image analysis further revealed that the larger BK-VLPs were composed of 72 capsomers which all were pentamers arranged in a T = 7 surface lattice. This system provides useful information for detailed studies of viral morphogenesis and the structural basis for the antigenicity of BKV.

Amino Acid Sequence↗
PubMed2003

Molecular investigation of hepatitis E virus infection in patients with acute hepatitis in Kathmandu, Nepal.

One hundred fifty-four consecutive patients with sporadic acute hepatitis, who were seen at a city hospital in the Kathmandu valley of Nepal in 1997, were studied. IgM antibodies to hepatitis A virus were detected in four patients (3%), IgM antibodies to hepatitis B core in four patients (3%), hepatitis B surface antigen in 20 (13%), and hepatitis C virus RNA in four patients (3%). IgM antibodies to hepatitis E virus (HEV) (anti-HEV IgM) and HEV RNA were detected in 77 (50%) and 48 (31%), respectively. Consequently, 86 patients (56%) including nine HEV-viremic patients without anti-HEV IgM, were diagnosed with hepatitis E. The cause of hepatitis was not known in 53 patients (34%). All 48 HEV RNA-positive samples were genotyped as 1, and subtyped further as 1a in 17 (35%), 1c in 29 (60%), and mixed infection of 1a and 1c in 2 (4%). A seasonal difference in the prevalence of HEV subtypes was recognized. Before the rainy season (January to July), both 1a and 1c isolates were found: the intrasubtypic difference was up to 9.0% and 1.7%, respectively, in the 412-nucleotide sequence of open reading frame 2. During the rainy season (August), only 1c isolates (n = 17) with 99.5-100% identity were found; 13 of 17 isolates had the same sequence, being identical to the 3 isolates that emerged at the end of July. These results suggest that a particular HEV 1c strain spread widely during the rainy season and was implicated in a small epidemic in the Kathmandu valley in August 1997.

Acute Disease↗
PubMed2003

Evidence for widespread infection of hepatitis E virus among wild rats in Japan.

Sporadic cases of hepatitis E have been reported in industrialized countries, including Japan. The source of hepatitis E virus (HEV) in these patients is unknown, although zoonotic transmission has been suggested. To investigate whether or not rodents might be a reservoir of HEV, we conducted an epidemiological survey for the antibody to a recombinant capsid protein of HEV using serum samples from wild rodents in Japan. One hundred and fourteen of 362 (31.5%) Norway rats (Rattus norvegicus) and 12 of 90 (13.3%) black rats (Rattus rattus) were positive for anti-HEV IgG. In contrast, all of the sera from 55 mice were negative for anti-HEV IgG. The rate of antibody positivity increased with weight among Norway rats. Seropositive rats were found in all five districts surveyed in this study, but the prevalence of anti-HEV IgG in wild rats differed among these prefectures. Despite the fact that Japan is a non-endemic country of hepatitis E, widespread infection of HEV was observed among wild rats in Japan. Our results suggested that HEV or a closely related virus is circulating among wild rats in Japan.

Journal Article↗
PubMed2003

Broadly reactive and highly sensitive assay for Norwalk-like viruses based on real-time quantitative reverse transcription-PCR.

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.

Base Sequence↗
PubMed2003

Prevalence of antibody against hepatitis E virus in various species of non-human primates: evidence of widespread infection in Japanese monkeys (Macaca fuscata).

We screened 495 serum samples from 20 species of non-human primates for the antibody against hepatitis E virus (HEV). Anti-HEV IgG was detected in 84 of 232 (36.2%) Japanese monkeys, 2 of 19 (10.5%) cynomolgus monkeys, 3 of 83 (3.6%) rhesus monkeys, and 1 of 1 (100%) Taiwanese monkey, respectively. These results suggest that HEV is circulating among monkeys belonging to the genus macaca. A high prevalence of anti-HEV IgG was observed in Japanese macaques (M. fuscata) despite the fact that Japan is non-endemic for hepatitis E. It is possible that HEV can be transmitted from Japanese macaques to humans. Further, the rate of antibody positivity was found to increase with age in Japanese macaques. Seropositive macaques were found throughout Japan, but the seroprevalence rate differed among geographic regions.

Animals↗
PubMed2003

[Hepatitis E].

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Animals↗
PubMed2002

Phylogenetic analysis of the complete genome of 18 Norwalk-like viruses.

"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.

Adult↗
PubMed2002

Molecular diagnosis of human enteroviruses by phylogeny-based classification by use of the VP4 sequence.

Human enteroviruses (EVs) are the major cause of a variety of acute and chronic illnesses. Virus isolation and neutralization tests are usually done to identify the causative virus, but these tests are labor intensive, time consuming, and sometimes require suckling mice from which certain viruses have been isolated. This study investigated a rapid and reliable method based on reverse-transcription polymerase chain reaction and phylogenetic analysis. The phylogenetic tree constructed by neighbor-joining on the basis of the VP4 sequence from 66 prototypes grouped all human EVs into 5 distinct clusters. These clusters correspond closely to the 5 newly designated species-human EV A-D and poliovirus. The VP4 sequences of 89 isolates from 26 serotypes obtained over >30 years plus those of 66 prototype strains were analyzed. Each isolate formed a monophyletic cluster along with its respective prototype strain, allowing for serotype identification (with the exception of E-8). VP4-based classification appears to be an effective tool for the molecular epidemiology study of EVs.

Capsid↗
PubMed2002

Chimeric recombinant hepatitis E virus-like particles as an oral vaccine vehicle presenting foreign epitopes.

Many viral and bacterial pathogens establish infections through mucosal surfaces in their initial stage. However, only a few nonreplicating molecules successfully induce strong mucosal immune reaction without the addition of adjuvants by oral administration. To overcome this difficulty, we investigated whether hepatitis E virus-like particles (HEV-VLPs) could be utilized as a carrier molecule for foreign antigenic epitopes and to stimulate mucosal immunity without the need for adjuvants. To accomplish this goal, we incorporated a B cell epitope tag, consisting of 11 amino acids at the C-terminal of HEV-VLP. The chimeric VLP showed morphology similar to that of the mature HEV virion and VLP. The inserted epitope was reactive with a specific monoclonal antibody in the VLP form, suggesting that it was exposed on the surface of the VLP. After oral administration without adjuvant, this chimeric HEV induced significant levels of specific IgG and IgA to both the inserted epitope and HEV-VLP in intestinal secretions. These humoral immune responses were observed as early as 2 weeks after the first immunization. These results suggest the potential of HEV-VLP as a mucosal vaccine carrier vehicle for the presentation of antigenic epitopes through oral administration.

Administration, Oral↗
PubMed2002

Application of an automated specimen search system installed in a transmission electron microscope for the detection of caliciviruses in clinical specimens.

To evaluate the performance of an automated specimen search system in the detection of caliciviruses such as Norwalk-like viruses and Sapporo-like viruses, a suitable negative staining method was developed and the viruses were examined using the system installed in a transmission electron microscope (TEM). Clear images of the viruses were obtained by staining with 2% uranyl acetate at pH 4.0 as compared with 2% phosphotungstic acid staining at any pH. When the image parameter of 30+/-6 nm for the diameter of a single virus-like particle of 2% uranyl-acetate-stained Norwalk-like virus was set on the automated specimen search system, 95% of the virus-like particles that were counted by the conventional TEM technique were detected. The system was used to detect Norwalk-like viruses in five semipurified stool samples in which Norwalk-like viruses had already been detected by reverse transcription-polymerase chain reaction assay and conventional electron microscopy. The positive detection rate for Norwalk-like viruses, which had been counted by the conventional technique, ranged from 56.2 to 77.9% using this system. Our findings indicate that the automated specimen search system installed in a TEM is suitable for the detection of caliciviruses in semipurified stool samples. The system is useful for clinical diagnosis without the need for operator intervention.

Automation↗
PubMed2002

Genogroup-specific PCR primers for detection of Norwalk-like viruses.

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.

Base Sequence↗
PubMed2002

Cross-reactivity among several recombinant calicivirus virus-like particles (VLPs) with monoclonal antibodies obtained from mice immunized orally with one type of VLP.

Human caliciviruses (HuCVs) are classified into the Norwalk-like viruses (NLV) and Sapporo-like viruses (SLV) as genera within the family CALICIVIRIDAE: The NLV genus is further classified into genogroups I and II, based on sequence similarities. To study the antigenic determinants on the HuCV capsid protein and develop new diagnostic tools for field samples, we established and characterized monoclonal antibodies (MAbs) against baculovirus-expressed recombinant HuCV virus-like particles (VLPs). Hybrid clones producing MAbs were obtained from cultures of PAI myeloma cells fused with spleen or mesenteric lymph node cells from mice immunized orally with either a single type of recombinant Norwalk virus (rNV), Kashiwa 47 virus (rKAV), Snow Mountain agent (rSMA), or Sapporo virus (rSV) VLP or with mixtures of two types of VLPs from different genogroups. Twenty MAbs, obtained as mouse ascites, were characterized and classified into six groups according to their enzyme-linked immunosorbent assay (ELISA) and Western blotting (WB) cross-reactivity patterns to VLPs. Five groups of MAbs reacted by both WB and ELISA and were classified as follows: common cross-reactive MAbs for four genogroup I and six genogroup II VLPs (group A), genogroup I-specific MAbs (group B), genogroup II-specific MAbs (group C), and strain-specific MAbs (groups D and E). One MAb group (group F) reacted only by ELISA. The group A MAbs, which showed broad cross-reactivity with VLPs of both NLV genogroups, were obtained from mice immunized orally with a single type of VLP (either rNV or rKAV). Two MAbs, which were obtained from mice immunized with rSV, reacted with rSV but not with any NLV VLP. These are the first MAbs to be reported for any SLV. These strain-, genogroup-, and genus-reactive MAbs will be useful tools for further study of the antigenic and structural topography of the HuCV virion and for diagnostic assays for HuCVs.

Animals↗
PubMed2002

Identification of active-site amino acid residues in the Chiba virus 3C-like protease.

The 3C-like protease of the Chiba virus, a Norwalk-like virus, is one of the chymotrypsin-like proteases. To identify active-site amino acid residues in this protease, 37 charged amino acid residues and a putative nucleophile, Cys139, within the GDCG sequence were individually replaced with Ala in the 3BC precursor, followed by expression in Escherichia coli, where the active 3C-like protease would cleave 3BC into 3B (VPg) and 3C (protease). Among 38 Ala mutants, 7 mutants (R8A, H30A, K88A, R89A, D138A, C139A, and H157A) completely or nearly completely lost the proteolytic activity. Cys139 was replaceable only with Ser, suggesting that an SH or OH group in the less bulky side chain was required for the side chain of the residue at position 139. His30, Arg89, and Asp138 could not be replaced with any other amino acids. Although Arg8 was also not replaceable for the 3B/3C cleavage and the 3C/3D cleavage, the N-terminal truncated mutant devoid of Arg8 significantly cleaved 3CD into 3C and 3D (polymerase), indicating that Arg8 itself was not directly involved in the proteolytic cleavage. As for position 88, a positively charged residue was required because the Arg mutant showed significant activity. As deduced by the X-ray structure of the hepatitis A virus 3C protease, Arg8, Lys88, and Arg89 are far away from the active site, and the side chain of Asp138 is directed away from the active site. Therefore, these are not catalytic residues. On the other hand, all of the mutants of His157 in the S1 specificity pocket tended to retain very slight activity, suggesting a decreased level of substrate recognition. These results, together with a sequence alignment with the picornavirus 3C proteases, indicate that His30 and Cys139 are active-site residues, forming a catalytic dyad without a carboxylate directly participating in the proteolysis.

3C Viral Proteases↗
PubMed2002

Human rhinovirus 87 identified as human enterovirus 68 by VP4-based molecular diagnosis.

Human rhinoviruses (HRVs) are the major cause of respiratory infections. We developed a diagnostic method for HRVs based on the reverse-transcription polymerase chain reaction (RT-PCR) and VP4-based phylogenetic analysis. A set of primers used in the RT-PCR of human enteroviruses (EVs) appeared to be capable of amplifying all prototype strains of HRVs, each of which generated a 530-bp fragment. The single exception was HRV-87, which generated a 650-bp fragment, as observed in human EVs. The VP4 nucleotide sequence of HRV-87 showed more than 97% nucleotide identity with human EV-68, and formed a monophyletic cluster along with the prototype strain of EV-68 in the human EV-D cluster. HRV-87 showed the second highest homology (76.8%) with EV-70, another member of the human EV-D, in a sample of 66 human EVs and 12 HRVs. Therefore, HRV-87 should be reclassified into the cluster containing human EV-68.

Capsid Proteins↗
PubMed2002

Hepatitis e virus infection in fulminant hepatitis patients and an apparently healthy population in Bangladesh.

This is the first study comparing hepatitis E virus (HEV) infection in Bangladesh in fulminant hepatitis (FH) patients presumed to have a viral cause and in the apparently healthy population. Sera from 22 FH patients were analyzed for antibodies to hepatitis A virus (HAV), hepatitis B virus (HBV), hepatitis C and D viruses, and HEV and for hepatitis B surface antigen (HBsAg). Anti-HEV immunoglobulin M (IgM) was detected in the sera of 63.6% of patients, whereas 35.7% were positive for HBsAg. A high prevalence of HEV infection (83.3%) was noted in the HBV carriers. Serum samples from 273 apparently healthy individuals were tested for antibodies to HAV and HEV. Anti-HEV IgM was detected in 7.3% of the samples. The seroprevalence of HAV differed from that of HEV in the same population because all samples were negative for anti-HAV IgM. These data indicate that HEV infection is highly endemic in Bangladesh.

Adolescent↗
PubMed2002

[Norwalk virus: recent findings].

Acute gastroenteritis due to Norwalk virus(NV), a member of the family Caliciviridae, is a common illness worldwide. NV is a human pathogen causing food-borne and water-borne diseases that occur in various epidemiological settings. The disease is mild and self-limiting and the symptomatic phase lasts 24 to 72 hours. Genetic analyses of the polymerase and the capsid protein indicate that NV is grouped into two clusters, genogroup I and genogroup II. The development of recombinant virus-like particles made a great impact on the development of diagnostic assays such as antigen ELISA and antibody ELISA. Strong control measures are needed to prevent oyster-related outbreaks.

Caliciviridae Infections↗
PubMed2002

[Phylogenetic analysis of 14 strains of Norwalk-like viruses: identification of the region in the genome for genotyping].

'Norwalk-like viruses(NLV)', a member of the family Caliciviridae, are the major causative agent of acute gastroenteritis and genetically divided into two groups, geno-group I(GI) and genogroup II(GII). We have determined complete nucleotide sequences, of 9 new NLV strains. Using this information together with 5 known NLV sequences, we investigated the criteria to further classify genotypes of NLV. Validation of the topological error based on the bootstrap value and the branch length(distance) identified two potential subgenomic regions suitable for genotyping. They were the RNA dependent RNA polymerase(RdRp) and the capsid N-terminal/Shell domains(capsid N/S). When the distance distribution analysis was performed, the RdRp-based classification did not separate the strains into internal clusters within genogroup. Furthermore, a diversity plot analysis of the complete nucleotide sequences of the GII Saitama U1 strain indicated that the genotype was different between the RdRp and the capsid N/S, suggesting that these strains are the genetic recombinants. Therefore, RdRp is not suitable for genotyping. On the other hand, the clustering based on the capsid N/S successfully distinguished the NLV equally to the cluster according to the antigenicity determined by both antigen and antibody ELISAs with recombinant virus-like particles. We would like to recommend to use the capsid N/S for the genotyping.

Base Sequence↗