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Biomedical subjects

Ikuo Takashima

Publications and source records attributed to Ikuo Takashima.

At least 19 recordsLinked to original sources
PubMed2006

The kinetics of proinflammatory cytokines in murine peritoneal macrophages infected with envelope protein-glycosylated or non-glycosylated West Nile virus.

The envelope (E) protein glycosylation status of the New York strain of West Nile (WN) virus is an important determinant of virus neuroinvasiveness. To elucidate the determinant of the difference between E protein-glycosylated and non-glycosylated WN virus infections, the cytokine expression of murine peritoneal macrophages infected with each virus was examined. Tumor necrosis factor (TNF) alpha and interleukin (IL)-1beta were up-regulated with replication of the E protein-glycosylated virus. Interferon (IFN) beta and IL-6 were up-regulated with the clearance of both viruses. These results suggest that TNFalpha and IL-1beta expression are related to the virulence of E protein-glycosylated WN virus.

Animals↗
PubMed2006

Development of an enzyme-linked immunosorbent assay for serological diagnosis of tick-borne encephalitis using subviral particles.

The similarity of symptoms produced by tick-borne encephalitis (TBE) and Japanese encephalitis (JE) and the high degree of cross-reactivity between TBE and JE viruses by serological tests make the development of a differential diagnostic test a priority. In this study, recombinant prM/E proteins of TBE virus strain Oshima 5-10 expressed in mammalian cells resulted in the release of subviral particles (SPs) into the culture medium. Using the SPs as antigens, enzyme-linked immunosorbent assay (ELISA) systems were developed to detect TBE virus-specific IgM and IgG antibodies, designated SP-IgG and SP-IgM ELISAs, respectively. Of 83 serum samples from encephalitis patients in Khabarovsk, Russia, which were positive with the neutralization test (NT), 82 were positive by the SP-IgG ELISA, for a sensitivity of 98.8%, which was higher than that of a commercial ELISA kit. All 12 NT-negative samples were also negative by the SP-IgG ELISA (specificity, 100%). Of 17 patient samples that were NT-positive, 16 (94.1%) were positive by the SP-IgM ELISA. Of 15 paired serum samples that yielded equivocal results by NT, 11 had positive results with the SP-IgM ELISA, indicating a diagnosis of TBE infection. The SP-IgG and SP-IgM ELISAs showed no cross-reactivity with antibodies to the JE virus. The results indicate that these ELISAs will be useful for the detection of TBE-specific antibodies.

Animals↗
PubMed2006

Soochong virus: an antigenically and genetically distinct hantavirus isolated from Apodemus peninsulae in Korea.

Hantaan (HTN) virus, the etiologic agent of clinically severe hemorrhagic fever with renal syndrome (HFRS), was first isolated in 1976 from lung tissue of a striped-field mouse (Apodemus agrarius) captured in Songnae-ri, Gyeonggi Province, Republic of Korea. Found primarily in mountainous areas, the Korean field mouse (A. peninsulae) is the second-most dominant field rodent species found throughout Korea. A new hantavirus, designated Soochong (SOO), was isolated in Vero E6 cells from four A. peninsulae captured in August 1997 at Mt. Gyebang in Hongcheon-gun, Mt. Gachil, Inje-gun, Gangwon Province, and in September 1998 at Mt. Deogyu, Muju-gun, Jeollabuk Province. The entire S, M, and L genomic segments of SOO virus, amplified by RT-PCR from lung tissues of seropositive A. peninsulae and from virus-infected Vero E6 cells, diverged from HTN virus (strain 76-118) by 15.6%, 22.8%, and 21.7% at the nucleotide level and 3.5%, 9.5%, and 4.6% at the amino acid level, respectively. Phylogenetic analyses of the nucleotide and deduced amino acid sequences, using the maximum parsimony and neighbor-joining methods, indicated that SOO virus was distinct from A. agrarius-borne HTN virus. SOO virus shared a common ancestry with Amur virus from Far East Russia, as well as with H5 and B78 hantaviruses, previously isolated from HFRS patients in China. Cross-focus-reduction neutralizating antibody tests showed that SOO virus, which is the first hantavirus isolated in cell culture from A. peninsulae, could be classified as a new hantavirus serotype.

Animals↗
PubMed2006

Cytokine regulation in SARS coronavirus infection compared to other respiratory virus infections.

The pathogenesis of severe acute respiratory syndrome (SARS) is poorly understood and cytokine dysregulation has been suggested as one relevant mechanism to be explored. We compared the cytokine profile in Caco2 cells after infection of SARS coronavirus (SARS-CoV) with other respiratory viruses including respiratory syncytial virus (RSV), influenza A virus (FluAV), and human parainfluenza virus type 2 (hPIV2). Interferon (IFN) system (production and response) was not suppressed by SARS-CoV infection. Therefore, SARS-CoV replication was suppressed by pretreatment with IFN. SARS-CoV and RSV induced high levels of IL-6 and RANTES compared with FluAV and hPIV2. Induction level of suppressor of cytokine signaling-3 (SOCS3) by SARS-CoV was significantly lower than that by RSV in spite of the significant production of IL-6. Toll-like receptors 4 and 9, which correlate with the induction of inflammatory response, were upregulated by SARS-CoV infection. Collectively, overinduction of inflammatory cytokine and dysregulation of cytokine signaling may contribute to the immunopathology associated with "severe" inflammation in SARS.

Caco-2 Cells↗
PubMed2006

Inactivation of SARS coronavirus by means of povidone-iodine, physical conditions and chemical reagents.

The efficacy of several povidone-iodine (PVP-I) products, a number of other chemical agents and various physical conditions were evaluated for their ability to inactivate the severe acute respiratory syndrome coronavirus (SARS-CoV). Treatment of SARS-CoV with PVP-I products for 2 min reduced the virus infectivity from 1.17 x 10(6) TCID(50)/ml to below the detectable level. The efficacy of 70% ethanol was equivalent to that of PVP-I products. Fixation of SARS-CoV-infected Vero E6 cells with a fixative including formalin, glutaraldehyde, methanol and acetone for 5 min or longer eliminated all infectivity. Heating the virus at 56 degrees C for 60 min or longer reduced the infectivity of the virus from 2.6 x 10(7) to undetectable levels. Irradiation with ultraviolet light at 134 microW/cm(2) for 15 min reduced the infectivity from 3.8 x 10(7) to 180 TCID(50)/ml; however, prolonged irradiation (60 min) failed to eliminate the remaining virus, leaving 18.8 TCID(50)/ml.

Acetone↗
PubMed2005

Role of the N-linked glycans of the prM and E envelope proteins in tick-borne encephalitis virus particle secretion.

The tick-borne encephalitis (TBE) virus has two membrane glycoproteins (prM and E), which each has one N-linked glycan. Constructs that express prM and E proteins of TBE virus have been shown to produce virus-like particles (VLPs), which have surface properties that are similar to those of infectious viruses. To reveal the function of glycosylation of the TBE virus prM and E proteins in the secretion of VLPs, we expressed glycosylation-mutated prM and E proteins and compared the secretion levels and biological properties of the VLPs. In the prM protein glycosylation-deficient mutant, the level of secreted E protein was reduced to 60% of the wild-type level. On the other hand, in the E or prM-E protein glycosylation-deficient mutant, the level of secreted E protein was reduced to 10% of the wild-type level. Furthermore, the mutant which was glycosylated at positions 66 and 154 in protein E, the level of secreted E protein was four-fold higher than that of the wild-type. However, in the mutant which was glycosylated at position 66 only, E protein secretion was reduced to only 10% of the wild-type level. These data suggest that the glycan associated with the N-linked glycosylation site at position 154 in protein E plays an important role in VLP secretion.

Encephalitis Viruses, Tick-Borne↗
PubMed2005

Packaging the replicon RNA of the Far-Eastern subtype of tick-borne encephalitis virus into single-round infectious particles: development of a heterologous gene delivery system.

The sub-genomic replicon of tick-borne encephalitis (TBE) virus (Far-Eastern subtype) was packaged into infectious particles by providing the viral structural proteins in trans. Sequential transfection of TBE replicon RNA and a plasmid that expressed the structural proteins led to the secretion of infectious particles that contained TBE replicon RNA. The secreted particles had single-round infectivity, which was inhibited by TBE virus-neutralizing antibody. The physical structure of the particles was almost identical to that of infectious virions, and the packaged replicon RNA showed no recombination with the mRNAs of the viral structural proteins. Furthermore, heterologous genes were successfully delivered and expressed by packaging TBE replicon RNA with inserted GFP and Neo genes. This replicon packaging system may be a useful tool for the molecular study of the TBE virus genome packaging mechanism, and for the development of vaccine delivery systems.

Animals↗
PubMed2005

Novel rapid immunochromatographic test based on an enzyme immunoassay for detecting nucleocapsid antigen in SARS-associated coronavirus.

A novel severe acute respiratory syndrome-associated coronavirus (SARS-CoV) has been discovered. The detection of both antigens and antibodies in SARS-CoV from human specimens with suspected SARS plays an important role in preventing infection. We developed a novel rapid immunochromatographic test (RICT) based on the sandwich format enzyme immunoassay (EIA) with an all-in-one device for detecting the native nucleocapsid antigen (N-Ag) of SARS-CoV using monoclonal antibodies (MoAbs), which we produced by immunizing recombinant N-Ag to mice. RICT is a qualitative assay for respiratory aspirates and serum specimens. With this assay, a positive result can be judged subjectively by the appearance of a blue line on the device 15 min after the sample is applied. RICT with several pairs of MoAbs showed a high sensitivity for the detection of recombinant N-Ag as well as viral N-Ag of SARS-CoV. rSN122 and rSN21-2 were the best MoAbs for immobilized antibody and enzyme labeling, respectively. With regard to analytical sensitivity, RICT detected N-Ag at 31 pg/mL for recombinant N-Ag, and at 1.99 x 10(2) TCID(50)/mL for SARS-CoV. The specificity of RICT was 100% when 150 human sera and 50 nasopharyngeal aspirates (NSPs) were used. RICT based on an EIA using the rSN122/rSN21-2 pair is a sensitive, specific, and reliable rapid assay for detecting N-Ag in SARS-CoV treated with either heat or Triton X-100.

Animals↗
PubMed2005

Detection of West Nile virus and Japanese encephalitis virus using real-time PCR with a probe common to both viruses.

A diagnostic method to distinguish between West Nile virus (WNV) and Japanese encephalitis virus (JEV) based on fluorogenic real-time polymerase chain reaction (TaqMan) assays was developed. To detect WNV and JEV with a single probe, a probe was designed to correspond to sequences in the core protein region that are shared by both viruses. The specificity of this assay depended on the primer sets used, which were specific to the target virus sequences: the primer set for WNV could detect only WNV strains and the primer set for JEV could detect only JEV strains. The assays were tested by detection of viruses from experimentally infected animal tissues. The method described in this study will be useful for the simultaneous discrimination of WNV and JEV in areas where JEV is endemic, such as East Asia.

Animals↗
PubMed2005

[Phylogenetic analysis and pathogenicity of tick-borne encephalitis virus from Japan and far-east Russia].

Phylogenetic analysis of tick-borne encephalitis (TBE) virus revealed that Hokkaido strain of TBE virus evolved several hundreds years ago in far-east Russia. TBE virus strains in Irkutsk area were identified as Siberian subtype of TBE virus. BHK-cell adapted mutant of TBE virus showed lower neuro-invasive virulence in mice than parent virus. The mutant carried one amino acid substitution in envelope protein which resulted in increase of positive charge of the protein. The mutant-infected mice showed lower virus titers in bloods and spleens than the parent-infected mice. Infectious c-DNA clone of TBE virus Hokkaido strain was successfully generated and was applied to examine the neurovirulence in mice. One amino acid change in envelope protein and 2 amino acid changes in Ns5 protein showed a synergistic effect on reduced neurovirulence in mice.

Amino Acid Substitution↗
PubMed2005

[The present status and points of viral zoonoses].

In recent years, viral zoonoses are causing public health problems in Japan and abroad. This is due to the outbreaks of emerging viral zoonoses at different localities in the world. Due to the globalization, these emerging viral zoonoses may invade into Japan. To prevent the invasion and spread of the emerging viral zoonoses, quarantine measures should be strengthened against movements of man and animals. Diagnostic tests and vaccines to the zoonoses should be developed. Epidemiological survey must be conducted against man and domestic and wild animals in the endemic regions. International cooperation and information exchange are essential to establish the preventive systems for the viral zoonoses.

Animals↗
PubMed2005

[Tick-borne encephalitis].

Tick-borne encephalitis (TBE) is viral zoonosis transmitted by the infected ticks harboring TBE virus. Mortality rate reaches 30% of in some cases. More than 10,000 patients were reported annually in Europe and Far-East Asia. In 1993, the first case of TBE was reported in Japan, and TBE virus was isolated, which showed TBE virus is endemic in Japan.

Animals↗
PubMed2004

A new model of Hantaan virus persistence in mice: the balance between HTNV infection and CD8(+) T-cell responses.

We established a viral persistence model that involves the adoptive transfer of spleen cells from immunocompetent mice (H-2(d)) into Hantaan virus (HTNV)-infected severe combined immunodeficient (SCID, H-2(d)) mice. The infection is maintained despite the presence of neutralizing antibodies, without apparent signs of disease, and there is a correlation between HTNV persistence and the lack of HTNV-specific CD8(+) T cells. In addition, disseminated HTNV infection before the initiation of immune responses appears to be important for virus persistence. The suppression of HTNV-specific CD8(+) T cells in the present model appears to occur at the periphery. The present study also demonstrates that CD8(+) T cells contribute to the clearance of HTNV. Thus, it seems that HTNV-specific CD8(+) T cells play a key role in HTNV persistence in mice. This model of viral persistence is useful for studies of immune responses and immunocytotherapy against viral infection.

Adoptive Transfer↗
PubMed2004

Different chemokine expression in lethal and non-lethal murine West Nile virus infection.

West Nile (WN) virus is a mosquito-borne flavivirus that can cause lethal encephalitis in humans and horses. The WN virus endemic in New York City (NY) in 1999 caused large-scale mortality of wild birds that was not evident in endemic areas in other parts of the world, and the pathogenesis of the WN virus strain isolated in NY (NY strain) appears to differ from that of previously isolated strains. However, the pathogenesis of NY strain infection remains unclear. This study examined CC (RANTES/CCL5, MIP-1 alpha/CCL3, MIP-1 beta/CCL4) and CXC (IP-10/CXCL10, B lymphocyte chemoattractant (BLC/CXCL13), and B cell- and monocyte-activating chemokine (BMAC/CXCL14)) chemokine expression during lethal NY strain and non-lethal Eg101 strain infection in mice. We found that the mRNA of the CC chemokines, RANTES, MIP-1 alpha, MIP-1 beta, and IP-10 was highly up-regulated in the brain of NY strain-infected mice. By contrast, BLC mRNA was not detected in either group of mice, and BMAC mRNA was highly up-regulated in late stage of infection with the non-lethal Eg101 strain relative to levels in NY strain-infected mice.

Animals↗
PubMed2004

Genetic and antigenic characterization of the Amur virus associated with hemorrhagic fever with renal syndrome.

The genetic and antigenic characteristics of the Amur (AMR) and Far East (FE) virus lineages, which are both within the genus Hantavirus, were studied. Representative viruses, H5 and B78 for AMR and Bao 14 for FE, were used. The entire small (S) and medium (M) segments, except for the 3'- and 5'-ends, were sequenced. The deduced amino acid sequences of AMR had 96.7 and 92.0-92.2% identities with the Hantaan (HTN) virus in the S and M segments, respectively. The amino acid sequences of FE had 99.1 and 97.9% identities in the S and M segments, respectively. The three viral strains and HTN virus had similar binding patterns to a panel of monoclonal antibodies (MAbs), except that one MAb did not bind AMR. However, sera from Apodemus peninsulae, naturally infected with AMR virus, neutralized homologous viruses at 1:160 to 1:320 dilutions and HTN at 1:20 to 1:40 dilutions. The anti-AMR serum neutralized homologous viruses at a 1:80 dilution and HTN at a 1:40 dilution. The anti-HTN serum did not neutralize AMR (<1:40 dilution), although it had a high neutralizing titer (1:320) against the homologous virus. Therefore, we suggest that AMR virus may constitute a distinct serotype within the genus Hantavirus.

Amino Acid Sequence↗
PubMed2004

Amino acid changes responsible for attenuation of virus neurovirulence in an infectious cDNA clone of the Oshima strain of tick-borne encephalitis virus.

A stable full-length infectious cDNA clone of the Oshima strain of Tick-borne encephalitis virus (Far-Eastern subtype) was developed by a long high-fidelity RT-PCR and one-step cloning procedure. The infectious clone (O-IC) had four amino acid substitutions and produced smaller plaques when compared with the parent Oshima 5-10 strain. Using site-directed mutagenesis, the substitutions were reverted to restore the parent virus sequence (O-IC-pt). Although genetically identical, parent virus Oshima 5-10 and virus recovered from O-IC-pt demonstrated some biological differences that are possibly explained by the presence of quasispecies with differing virulence characteristics within the original virus population. These observations may have implications for vaccines based on modified infectious clones. It was also demonstrated that the amino acid substitution E-S(40)-->P at position 40 in the envelope (E) glycoprotein was responsible for plaque size reduction, reduced infectious virus yields in cell culture and reduced mouse neurovirulence. Additionally, two amino acid substitutions in the non-structural (NS)5 protein (virus RNA-dependent RNA polymerase) NS5-V(378)-->A and NS5-R(674)-->K also contributed to attenuation of virulence in mice, but did not demonstrate a noticeable biological effect in baby hamster kidney cell culture. Comparative neurovirulence tests revealed how the accumulation of individual mutations (E-S(40)-->P, NS5-V(378)-->A and NS5-R(674)-->K) can result in the attenuation of a virus.

Amino Acid Sequence↗
PubMed2004

Single point mutation in tick-borne encephalitis virus prM protein induces a reduction of virus particle secretion.

Flaviviruses are assembled to bud into the lumen of the endoplasmic reticulum (ER) and are secreted through the vesicle transport pathway. Virus envelope proteins play important roles in this process. In this study, the effect of mutations in the envelope proteins of tick-borne encephalitis (TBE) virus on secretion of virus-like particles (VLPs), using a recombinant plasmid expression system was analysed. It was found that a single point mutation at position 63 in prM induces a reduction in secretion of VLPs. The mutation in prM did not affect the folding of the envelope proteins, and chaperone-like activity of prM was maintained. As observed by immunofluorescence microscopy, viral envelope proteins with the mutation in prM were scarce in the Golgi complex, and accumulated in the ER. Electron microscopic analysis of cells expressing the mutated prM revealed that many tubular structures were present in the lumen. The insertion of the prM mutation at aa 63 into the viral genome reduced the production of infectious virus particles. This data suggest that prM plays a crucial role in the virus budding process.

Animals↗