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Naokazu Takeda

Publications and source records attributed to Naokazu Takeda.

At least 19 recordsLinked to original sources
PubMed2006

Enhancement of sapovirus recombinant capsid protein expression in insect cells.

Human sapovirus (SaV) is uncultivable, but expression of the recombinant capsid protein (rVP1) in insect cells results in the formation of virus-like particles (VLPs) that are morphologically similar to the native viruses. However, the SaV rVP1 expression levels are considerably low. We have found that inclusions of short foreign nucleotide sequences inserted directly upstream from the predicted rVP1 AUG start codon lead to increased yield of VLPs. This method allowed us to express a SaV rVP1, which could not have been expressed to measurable or practical levels otherwise.

Animals↗
PubMed2006

Detection of human sapovirus by real-time reverse transcription-polymerase chain reaction.

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.

Australia↗
PubMed2006

Genetic and antigenic diversity among noroviruses.

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.

Amino Acid Sequence↗
PubMed2006

Identification of the cleavage sites of sapovirus open reading frame 1 polyprotein.

Sapovirus (SaV), a member of the family Caliciviridae, is a causative agent of acute gastroenteritis in humans and swine and is currently divided into five genogroups, GI-GV. The proteolytic processing of the SaV open reading frame 1 (ORF1) polyprotein with a human GII SaV Mc10 strain has recently been determined and the products are arranged in the following order: NH(2)-p11-p28-p35 (NTPase)-p32-p14 (VPg)-p70 (Pro-Pol)-p60 (VP1)-COOH. The cleavage site between p14 (VPg) and p70 (Pro-Pol) was identified as E(1055)/A(1056) by N-terminal amino acid sequencing. To identify other cleavage sites, a series of GII SaV Mc10 full-length clones containing disrupted potential cleavage sites in the ORF1 polyprotein were constructed and used to generate linear DNA templates for in vitro coupled transcription-translation. The translation products were analysed by SDS-PAGE or by immunoprecipitation with region-specific antibodies. N-terminal amino acid sequencing with Escherichia coli-expressed recombinant proteins was also used to identify the cleavage site between p32 and p14. These approaches enabled identification of the six cleavage sites of the Mc10 ORF1 polyprotein as E(69)/G(70), Q(325)/G(326), Q(666)/G(667), E(940)/A(941), E(1055)/A(1056) and E(1722)/G(1723). The alignment of the SaV full-length ORF1 amino acid sequences indicated that the dipeptides used for the cleavage sites were either E or Q at the P1 position and A, G or S at the P1' position, which were conserved in the GI, GII, GIII, GIV and GV SaV ORF1 polyprotein.

Binding Sites↗
PubMed2006

Virological, serological, and clinical features of an outbreak of acute gastroenteritis due to recombinant genogroup II norovirus in an infant home.

Norovirus (NV) is an important cause of acute nonbacterial gastroenteritis worldwide. Recently, several sporadic cases due to naturally occurring recombinant NVs have been reported. In January 2000, there was an outbreak of gastroenteritis in an infant home in Sapporo, Japan. Of 34 residents of the home that were less than 2 years old, 23 developed gastrointestinal symptoms and NV infection was confirmed by conventional reverse transcription-PCR to detect the RNA polymerase region of genogroup II NV. In this virus, the RNA polymerase region shared 86% nucleotide identity with Hawaii virus but only 77% with Mexico virus; however, its capsid region shared only 70% identity with Hawaii virus but 90% with Mexico virus. On the other hand, both regions shared a higher 96% nucleotide identity with Arg320 virus, which was found in Mendoza, Argentina, in 1995 and considered to be a recombinant of Hawaii and Mexico viruses. The findings indicate that the virus involved in the outbreak was similar and may have evolved from the Arg320 virus. Clinically the cases were more severe than those of previously reported sporadic or outbreak cases of NV infection.

Acute Disease↗
PubMed2006

Genetic diversity of Sapovirus in children, Australia.

Sapovirus was detected in 7 of 95 stool specimens from children with gastroenteritis of unknown etiology in Sydney, Australia, from August 2001 to August 2002 and from February 2004 to August 2004, by using reverse transcription-polymerase chain reaction. Sequence analysis of the N-terminal capsid region showed all human sapovirus genogroups.

Australia↗
PubMed2006

Serologic evidence for hepatitis E virus infection in mongoose.

Although pig and wild boar are considered to be the reservoirs of hepatitis E virus (HEV) in Japan, the spread of HEV to other animals is unknown. Serum samples from 84 mongooses (Small Asian mongoose; Herpestes javanicus) collected in Okinawa, Japan were examined for antibodies to HEV by enzyme-linked immunosorbent assay and RNA was analyzed by reverse transcription-polymerase chain reaction. Seven (8.3%) of 84 mongooses were positive for IgG antibodies to HEV, and the antibody-positive rate increased with body weight and size, whereas HEV RNA was not detected in these samples. These results are consistent with the possibility that mongooses in Okinawa are occasionally infected with HEV; however, they are not considered the major zoonotic reservoir of HEV.

Animals↗
PubMed2006

International collaborative survey on epidemiology of hepatitis E virus in 11 countries.

We conducted seroepidemiological studies on antibody prevalence to hepatitis E virus (HEV) in 5,233 sera from 11 countries to ascertain the present state of HEV infection on a global basis. The prevalence of anti-HEV IgG increased with age in these tested countries, but the rate of antibody positivity was over 20% in the 16-30 year-old group in most of the participating countries, except for Japan, the USA, and Spain. Of patients with acute hepatitis of unknown etiology from Nepal, 56% (14/25) were positive for the IgM class of anti-HEV antibody. In addition, HEV RNAs in the serum from 3 Nepali patients who had the IgM antibody were detected by nested PCR and all of the HEV genes isolated belonged to genotype 1. Our results indicate that HEV is spreading worldwide, not only in developing countries, but also in more industrialized countries than previously thought.

Adolescent↗
PubMed2006

Different fecal shedding patterns of two common strains of hepatitis E virus at three Japanese swine farms.

Zoonotic infections caused by eating the meat of deer, wild boar, and pig have been suggested in Japan, a country that is not epidemic for hepatitis E caused by hepatitis E virus (HEV). This virus is widely spread in domestic pigs in both epidemic and non-epidemic countries. We studied fecal HEV shedding patterns on three Japanese farms that had two common genotype III HEV strains. Two of the three farms had high shedding peaks (75% and 100%) in pigs 1-3 months of age, suggesting that these animals had the highest risk of spreading HEV through feces. Another farm had a low shedding rate in animals six months of age and a low prevalence of the IgG antibody to HEV. Fecal IgA antibody to HEV was found in sucking pigs < 13 days of age on farms that had high and low shedding patterns. A small fraction of pigs (3 of 43 [7%]) at the finishing stage (5-6 months of age) still shed HEV on the three farms.

Animals↗
PubMed2005

Challenges in creating a vaccine to prevent hepatitis E.

Recombinant hepatitis E virus capsid protein (HEV CP) assembles orally immunogenic virus-like particles (VLP) when expressed in an insect cell system. We used plant expression cassettes, pHEV101 and pHEV110, for transformation of potato to express HEV CP, and 10 independent transgenic lines of HEV101 and 6 lines of HEV110 were obtained. ELISA for HEV CP was performed on tuber extracts. Accumulation of HEV CP in tubers varied from about 5 to 30 microg/g fresh tuber depending on the transgenic plant line. We further compared the expression levels with the yield of tubers for each line. Tuber yield varied less than expression levels, and ranged from about 600 to 1000 g per pot. Although Western blot showed that apparently intact HEV CP accumulated, we observed very limited assembly of virus-like particles in potato tubers. Oral immunization of mice with transgenic potatoes failed to elicit detectable anti-CP antibody response in serum, suggesting that VLP assembly is a key factor in orally delivered HEV CP vaccines.

Animals↗
PubMed2005

Mutational study of sapovirus expression in insect cells.

Human sapovirus (SaV), an agent of human gastroenteritis, cannot be grown in cell culture, but expression of the recombinant capsid protein (rVP1) in a baculovirus expression system results in the formation of virus-like particles (VLPs). In this study we compared the time-course expression of two different SaV rVP1 constructs. One construct had the native sequence (Wt construct), whereas the other had two nucleotide point mutations in which one mutation caused an amino acid substitution and one was silent (MEG-1076 construct). While both constructs formed VLPs morphologically similar to native SaV, Northern blot analysis indicated that the MEG-1076 rVP1 mRNA had increased steady-state levels. Furthermore, Western blot analysis and an antigen enzyme-linked immunosorbent assay showed that the MEG-1076 construct had increased expression levels of rVP1 and yields of VLPs. Interestingly, the position of the mutated residue was strictly conserved residue among other human SaV strains, suggesting an important role for rVP1 expression.

Amino Acid Sequence↗
PubMed2005

Expression and antigenicity of virus-like particles of norovirus and their application for detection of noroviruses in stool samples.

Human noroviruses (NoVs), members of the genus Norovirus in the family Caliciviridae, are the leading agents of nonbacterial acute gastroenteritis worldwide. Human NoVs are currently divided into at least two genogroups, genogroup I (GI) and genogroup II (GII), each of which contains at least 14 and 17 genotypes. To explore the genetic and antigenic relationship among NoVs, we expressed the capsid protein of four genetically distinct NoVs, the GI/3 Kashiwa645 virus, the GII/3 Sanbu809 virus, the GII/5 Ichikawa754 virus, and the GII/7 Osaka10-25 virus in baculovirus expression system. An antigen enzyme-linked immunosorbent assay (ELISA) with hyperimmune serum against the four recombinant capsid proteins and characterized previously three capsid proteins derived from GI/1, GI/4, and GII/12 was developed to detect the NoVs antigen in stools. The antigen ELISA was highly specific to the homotypic strains, allowing assignment of a strain to a Norovirus genetic cluster within a genogroup.

Animals↗
PubMed2005

Characterization of the norovirus 3C-like protease.

The recombinant 3C-like protease of Chiba virus, a Norovirus, expressed in Escherichia coli cells was purified and characterized as to effects of pH, temperature, salt contents, and SH reagents on its proteolytic activity. The optimal pH and temperature of the 3C-like protease for the proteolytic activity were 8.6 and 37 degrees C, respectively. Increased concentration (approximately 100 mM) of monovalent cations such as Na+ and K+ was inhibitory to the activity. Hg2+ and Zn2+ remarkably inhibited the protease activity, while Mg2+ and Ca2+ had no virtual effect. Several sulfhydryl reagents such as p-chloromercuribenzoic acid, methyl methanethiosulfonate, N-ethylmaleimide and N-phenylmaleimide also blocked the activity, confirming the previous result that cysteine residue(s) were responsible for the proteolysis.

3C Viral Proteases↗
PubMed2005

Proteolytic processing of sapovirus ORF1 polyprotein.

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.

3C Viral Proteases↗
PubMed2005

Essential elements of the capsid protein for self-assembly into empty virus-like particles of hepatitis E virus.

Hepatitis E virus (HEV) is a noncultivable virus that causes acute liver failure in humans. The virus's major capsid protein is encoded by an open reading frame 2 (ORF2) gene. When the recombinant protein consisting of amino acid (aa) residues 112 to 660 of ORF2 is expressed with a recombinant baculovirus, the protein self-assembles into virus-like particles (VLPs) (T.-C. Li, Y. Yamakawa, K. Suzuki, M. Tatsumi, M. A. Razak, T. Uchida, N. Takeda, and T. Miyamura, J. Virol. 71:7207-7213, 1997). VLPs can be found in the culture medium of infected Tn5 cells but not in that of Sf9 cells, and the major VLPs have lost the C-terminal 52 aa. To investigate the protein requirement for HEV VLP formation, we prepared 14 baculovirus recombinants to express the capsid proteins truncated at the N terminus, the C terminus, or both. The capsid protein consisting of aa residues 112 to 608 formed VLPs in Sf9 cells, suggesting that particle formation is dependent on the modification process of the ORF2 protein. In the present study, electron cryomicroscopy and image processing of VLPs produced in Sf9 and Tn5 cells indicated that they possess the same configurations and structures. Empty VLPs were found in both Tn5 and Sf9 cells infected with the recombinant containing an N-terminal truncation up to aa residue 125 and C-terminal to aa residue 601, demonstrating that the aa residues 126 to 601 are the essential elements required for the initiation of VLP assembly. The recombinant HEV VLPs are potential mucosal vaccine carrier vehicles for the presentation of foreign antigenic epitopes and may also serve as vectors for the delivery of genes to mucosal tissue for DNA vaccination and gene therapy. The results of the present study provide useful information for constructing recombinant HEV VLPs having novel functions.

Animals↗
PubMed2005

A norovirus protease structure provides insights into active and substrate binding site integrity.

Norovirus 3C-like proteases are crucial to proteolytic processing of norovirus polyproteins. We determined the crystal structure of the 3C-like protease from Chiba virus, a norovirus, at 2.8-A resolution. An active site including Cys139 and His30 is present, as is a hydrogen bond network that stabilizes the active site conformation. In the oxyanion hole backbone, a structural difference was observed probably upon substrate binding. A peptide substrate/enzyme model shows that several interactions between the two components are critical for substrate binding and that the S1 and S2 sites appropriately accommodate the substrate P1 and P2 residues, respectively. Knowledge of the structure and a previous mutagenesis study allow us to correlate proteolysis and structure.

3C Viral Proteases↗
PubMed2005

Structure and assembly of a T=1 virus-like particle in BK polyomavirus.

In polyomaviruses the pentameric capsomers are interlinked by the long C-terminal arm of the structural protein VP1. The T=7 icosahedral structure of these viruses is possible due to an intriguing adaptability of this linker arm to the different local environments in the capsid. To explore the assembly process, we have compared the structure of two virus-like particles (VLPs) formed, as we found, in a calcium-dependent manner by the VP1 protein of human polyomavirus BK. The structures were determined using electron cryomicroscopy (cryo-EM), and the three-dimensional reconstructions were interpreted by atomic modeling. In the small VP1 particle, 26.4 nm in diameter, the pentameric capsomers form an icosahedral T=1 surface lattice with meeting densities at the threefold axes that interlinked three capsomers. In the larger particle, 50.6 nm in diameter, the capsomers form a T=7 icosahedral shell with three unique contacts. A folding model of the BKV VP1 protein was obtained by alignment with the VP1 protein of simian virus 40 (SV40). The model fitted well into the cryo-EM density of the T=7 particle. However, residues 297 to 362 of the C-terminal arm had to be remodeled to accommodate the higher curvature of the T=1 particle. The loops, before and after the C-terminal short helix, were shown to provide the hinges that allowed curvature variation in the particle shell. The meeting densities seen at the threefold axes in the T=1 particle were consistent with the triple-helix interlinking contact at the local threefold axes in the T=7 structure.

BK Virus↗