Biomedical subjects

Ikuo Takashima

Publications and source records attributed to Ikuo Takashima.

34 records · Page 2Linked to original sources

PubMed2004

Viral envelope protein glycosylation is a molecular determinant of the neuroinvasiveness of the New York strain of West Nile virus.

Two New York (NY) strains of the West Nile (WN) virus were plaque-purified and four variants that had different amino acid sequences at the N-linked glycosylation site in the envelope (E) protein sequence were isolated. The E protein was glycosylated in only two of these strain variants. To determine the relationship between E protein glycosylation and pathogenicity of the WN virus, 6-week-old mice were infected subcutaneously with these variants. Mice infected with viruses that carried the glycosylated E protein developed lethal infection, whereas mice infected with viruses that carried the non-glycosylated E protein showed low mortality. In contrast, intracerebral infection of mice with viruses carrying either the glycosylated or non-glycosylated forms of the E protein resulted in lethal infection. These results suggested that E protein glycosylation is a molecular determinant of neuroinvasiveness in the NY strains of WN virus.

Animals↗

PubMed2004

Epizootiological and epidemiological study of hantavirus infection in Japan.

Epizootiological surveys on hantavirus infections in rodents were carried out in various areas of Japan, including the four major islands of Hokkaido, Honshu, Shikoku, and Kyushu from 2000 to 2003. A total of 1,221 rodents and insectivores were captured. Seropositive animals were found in Apodemus (A.) speciosus (5/482, 1.0%), Rattus (R.) norvegicus (4/364, 1.1%), R. rattus (3/45, 6.7%), and Clethrionomys (C.) rufocanus (7/197, 3.6%). The partial S segment was amplified from one seropositive R. rattus captured at Hakodate. The nucleotide sequence showed 96% identity with the Seoul virus (SEOV) prototype strain SR-11. In addition, we conducted an epidemiological survey on human hantavirus infection in a high-risk population, the personnel of the Japan Ground Self-defense Force on Hokkaido. One out of 207 human blood samples was positive for anti-hantavirus antibody by IFA, ELISA, and WB analysis. The result of the serotype specific ELISA indicates that this individual acquired SEOV infection. This study indicates that A. speciosus, R. norvegicus, R. rattus, and C. rufocanus carry hantaviruses as the reservoir animals in Japan. Infected R. rattus and R. norvegicus in port areas could be the sources of human SEOV infection and a threat to travelers and individuals working in seaports.

Animals↗

PubMed2004

Epizootiologic survey for Babesia microti among small wild mammals in northeastern Eurasia and a geographic diversity in the beta-tubulin gene sequences.

We previously reported that small wild rodents in Japan harbor two types of novel Babesia microti-like parasites (designated as Hobetsu and Kobe types), but not the type commonly found in the northeastern United States (U.S. type) where human babesiosis is endemic. To determine whether these new types of parasites are distributed in places surrounding Japan, an epizootiologic survey was undertaken in three geographically distant areas in northeastern Eurasia; South Korea, Vladivostok in Russia, and Xinjiang in China. Blood samples were collected from a total of 387 animals comprising 24 species. DNAs extracted from the samples were tested by nested PCR targeting babesial nuclear small-subunit rRNA gene (rDNA), which revealed that small rodents harboring B. microti exist in all three survey areas. Sequence analysis showed that all PCR-positive samples had rDNA sequences virtually identical to that of U.S.-type B. microti. However, when beta-tubulin gene sequences were compared, evident geographic variations were seen. By use of primers specific for each of the beta-tubulin genes of Kobe-, Hobetsu-, and U.S.-type parasites, a type-specific PCR was developed. Parasite with Hobetsu- or Kobe-type sequence was not detected from any of the three survey areas. These findings suggest that U.S.-type B. microti is widely distributed among small wild mammals in temperate zones of not only North America, but also Eurasia, whereas that Hobetsu- and Kobe-type parasites may be uniquely distributed in Japan.

Animals↗

PubMed2004

Comparison of virulence of various hantaviruses related to hemorrhagic fever with renal syndrome in newborn mouse model.

The virulence of hantaviruses that are antigenically related but have different genetic characteristics from the prototype of hantavirus, Hantaan (HTN) virus, was examined in newborn mice. The H5 and B78 strains of the Amur (AMR) genotype, the Bao14 strain of the Far East (FE) genotype, and the 76-118 strain of HTN virus were inoculated subcutaneously (1focus-forming unit; FFU) into newborn mice. All of the AMR and FE genotype viruses inoculated mice were died by 16 days post-infection (dpi) and 21 dpi, respectively, while 50% of the HTN virus inoculated mice survived until 30 dpi. The AMR and FE genotype viruses inoculated mice had high viral titers in the lung (1.3x10(6) to 1.3x10(8) FFU/gram [g] tissue) , brain (2.1x10(7) to 1.2x10(9) FFU/g tissue), and kidney(2.5x10(5) to 1.6x10(7) FFU/g tissue), and showed a detectable level of antibodies (titers 1:16-1:32) at 14 dpi. In contrast, the HTN virus infected mice had viruses only in the lungs at low titers (1.1-5.3x10(5) FFU/g tissue). Observations of body-weight changes revealed that the AMR and FE genotype viruses inoculated mice had lower growth rates than the HTN virus inoculated mice. These data suggest that the AMR and FE genotype viruses are more virulent than the HTN virus in newborn mice.

Animals↗

PubMed2004

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) TCID50/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 5 min dramatically reduced the infectivity of the virus from 2.6 x 10(7) to 40 TCID50/ml, whereas heating the virus for 60 min or longer eliminated all infectivity. Irradiation with ultraviolet light at 134 microW/cm2 for 15 min reduced the infectivity from 3.8 x 10(7) to 180 TCID50/ml; however, prolonged irradiation (60 min) failed to eliminate the remaining virus, leaving 18.8 TCID50/ml. We believe that these findings will be useful for the implementation of infection control measures against SARS, and for the establishment of effective guidelines for the prevention of SARS outbreaks.

Animals↗

PubMed2003

A BHK-21 cell culture-adapted tick-borne encephalitis virus mutant is attenuated for neuroinvasiveness.

We derived the baby hamster kidney (BHK)-21 cell culture-adapted, tick-borne encephalitis (TBE) virus mutant. To reveal the pathogenicity of the TBE virus, we compared the pathogenicity of the mutant (Oshima Cl-1) and parental (Oshima 5-10) virus in mouse model. The neurovirulence of mutant in mice was identical to that of parent. However, the level of neuroinvasiveness was higher for parent than for mutant. The degrees of viremia and virus titers in the spleen were lower in mice that were inoculated subcutaneously (s.c.) with mutant than in mice that received parent. Unlike parent, mutant was rarely detected in the brains of s.c. inoculated mice. Genetic analysis revealed that mutant had single amino acid substitutions in each of the E and NS5 proteins compared with parent. Furthermore, while mutant infection of BHK-21 cells was inhibited by glycosaminoglycans (GAGs), this was not the case for parent. In summary, the BHK-21-cell-adapted mutant virus showed reduced neuroinvasiveness in mice due to low-level induction of viremia. The attenuation process involved a single amino acid change in the E protein, which may have resulted in the rapid clearance of the virus due to its high affinity for negatively charged molecules in vivo.

Amino Acid Sequence↗

PubMed2003

Enzyme-linked immunosorbent assay using recombinant antigens expressed in mammalian cells for serodiagnosis of tick-borne encephalitis.

A recombinant plasmid that expresses the tick-borne encephalitis (TBE) virus premembrane (prM) and envelope (E) proteins in mammalian cells was constructed. Recombinant proteins retained antigenic and conformational structures similar to those of native virus proteins, and transfected cells released virus-like particles (VLPs), which were 1.13-1.14 g/ml in density and 20-30 nm in diameter, into the culture medium. Recombinant E proteins were used for the development of an enzyme-linked immunosorbent assay (ELISA) to detect TBE virus-specific IgM and IgG antibodies in serum. The results of this ELISA correlated well with the results of commercial ELISA, when tested with 95 serum samples from clinically TBE-suspected patients. In addition, ELISA using recombinant antigens showed no cross-reactivity against serum from Japanese encephalitis (JE) patients, despite the cross-reactivity shown by commercial ELISA systems. These observations indicated that this newly developed ELISA system could distinguish tick-borne encephalitis from Japanese encephalitis infection, and that it constitutes a useful and safe alternative to conventional ELISA systems.

Animals↗

PubMed2003

Discrimination of West Nile virus and Japanese encephalitis virus strains using RT-PCR RFLP analysis.

West Nile (WN) virus is a mosquito-borne flavivirus that induces lethal encephalitis in humans and horses. Since an outbreak of WN encephalitis in humans and horses occurred in New York City in late August 1999, the possibility exists that WN virus will invade regions that have close links with the United States, such as Japan. We developed a genetic diagnostic method that discriminates between strains of WN virus and Japanese encephalitis (JE) virus. The method involves RT-PCR restriction fragment length polymorphism (RFLP) analysis with a RT-PCR primer set, a nested PCR primer set, and a restriction enzyme. We detected WN and JE viruses in experimentally infected animal brain, spleen, and serum samples. Our method is useful in distinguishing WN viruses from the endemic background of JE viruses, and in discriminating the highly virulent WN strain, which was isolated in New York in 1999, from other WN virus strains.

Animals↗

PubMed2003

Hantavirus-specific CD8(+)-T-cell responses in newborn mice persistently infected with Hantaan virus.

The relationship between virus-specific CD8(+)-T-cell responses and viral persistence was studied in mice by using Hantaan virus (HTNV). We first established a simple method for measuring levels of virus-specific CD8(+) T cells by flow cytometry. Next, to produce a mouse model of persistent HTNV infection, newborn mice were inoculated subcutaneously within 24 h of birth with 1 or 0.1 50% newborn mouse lethal dose of HTNV. All mice that escaped lethal infection were persistently infected with HTNV until at least 30 days after virus inoculation and had no virus-specific CD8(+) T cells producing gamma interferon (IFN-gamma). Subsequently, the virus was eliminated from some of the mice, depending on the appearance of functional virus-specific CD8(+) T cells, which have the ability to produce IFN-gamma and tumor necrosis factor alpha (TNF-alpha) and have cytotoxic activity. Neutralizing antibodies were detected in all mice, regardless of the presence or absence of virus. In the acute phase, which occurs within 30 days of infection, IFN-gamma-producing HTNV-specific CD8(+) T cells were detected on day 15 after virus inoculation. However, TNF-alpha production and the cytotoxic activity of these specific CD8(+) T cells were impaired and HTNV was not removed. Almost all of these specific CD8(+) T cells disappeared by day 18. These results suggest that functional HTNV-specific CD8(+) T cells are important for clearance of HTNV.

Animals↗

PubMed2003

Development of an efficient method for recovery of Puumala and Puumala-related viruses by inoculation of Mongolian gerbils.

Puumala (PUU) virus and PUU-related viruses are difficult to isolate in cell culture. To determine whether animal inoculation would be a better alternative for virus recovery, the Sotkamo strain of PUU virus was inoculated into several animal species. Newborn Mongolian gerbils (MGs), mice, and rats were infected with the Sotkamo strain by intracerebral (ic), intraperitoneal (ip), and subcutaneous (sc) inoculation. Antibodies to PUU appeared in MGs at 30 days post-infection (dpi), and in mice and rats at 15 dpi. Interestingly, virus appeared at 7 dpi in lung and brain of MGs inoculated via ic and ip routes. Virus was detected in all tested tissues of MGs at 15 dpi, with a peak level of 1.36 x 10 (5) focus forming units (FFU)/g in brain tissue. The virus titer declined with the onset of the antibody response and became undetectable by 75 dpi, when the antibody titer reached the maximum level. The appearance of the virus in mice and rats was delayed as compared to MGs, and the virus titer was apparently lower, at approximately 4 to 8 x 10(3) FFU/g, at 15 dpi. In addition, lung homogenates of antibody-positive Clethrionomys (C.) rufocanus (captured in Tobetsu, Hokkaido, Japan) were inoculated into MGs by the ic route. PUU-related viral RNA was detected at 16 dpi in the brains of MG inoculated with the lung homogenate, and antibodies were detected at 45 dpi. These findings indicate that newborn MG inoculation is an efficient method to recover PUU and PUU-related viruses.

Animals↗

PubMed2003

Establishment of an enzyme-linked immunosorbent assay for detection of hantavirus antibody of rats using a recombinant of nucleocapsid protein expressed in Escherichia coli.

A recombinant nucleocapsid protein of Hantaan virus (HTN) 76-118 strain expressed in E. coli was applied as a serodiagnostic antigen in an enzyme-linked immunosorbent assay (rHTN-ELISA) for detection of hantavirus antibody in rat sera. The sensitivity and specificity of the rHTN-ELISA were compared with those of the indirect immunofluoresent assay (IFA) using virus-infected cells. The sensitivity of rHTN-ELISA was similar to that of the IFA both in experimentally SR-11 infected rat and naturally infected rat sera. Sera showing a low antibody titer in IFA and suspected to be negative by other methods were also found to be negative in rHTN-ELISA. These results indicate that rHTN-ELISA is effective as a screening method for serodiagnosis of hantaviruses, because of its high sensitivity, specificity, safety and suitability for processing large number of samples.

Animals↗

PubMed2002

Evaluation of enzyme-linked immunosorbent assay (ELISA) and immunofluorescent antibody (IFA) test for the detection of porcine reproductive and respiratory syndrome virus (PRRSV) antibody in pigs from conventional farms.

To evaluate the immunofluorescent antibody (IFA) test and enzyme-linked immunosorbent assay (ELISA) for detecting the porcine reproductive and respiratory syndrome virus (PRRSV) antibody, conventional pigs in PRRSV-positive and -negative commercial farms were examined. Antibody development patterns in ELISA and IFA tests were compared in 3 week old piglets experimentally infected with the PRRSV. The virus was detected from 2 days post infection (PI) and then the antibody titers and S/P ratios rose by both methods. A total of 208 serum samples were collected from 4 PRRSV-negative farms and 210 samples from PRRSV-positive farms, and were tested for the PRRSV antibody by IFA and ELISA. The titer of 64 should be set as the cut-off point in IFA for field sera. Similarly, the cut-off S/P ratio should be set at 0.4 in ELISA. A high degree of correlation was observed between antibody titers by the two methods in these 418 samples, with a correlation coefficient of 0.84. The coincidence rate between the two tests was 84.7% (354/418). In non-coincident cases, ELISA was able to detect the antibody with a low titer in the serum samples which were negative in IFA but from PRRSV positive farms. ELISA was more sensitive than IFA to detect PRRSV infected animals or farms.

Animals↗

PubMed2002

Genetic characterization of hantaviruses transmitted by the Korean field mouse (Apodemus peninsulae), Far East Russia.

In an epizootiologic survey of 122 rodents captured in Vladivostok, Russia, antibodies positive for hantavirus were found in Apodemus peninsulae (4/70), A. agrarius (1/39), and Clethrionomys rufocanus (1/8). The hantavirus sequences identified in two seropositive A. peninsulae and two patients with hemorrhagic fever with renal syndrome (HFRS) from the Primorye region of Far East Russia were designated as Solovey and Primorye, respectively. The nucleotide sequences of the Solovey, Primorye, and Amur (obtained through GenBank) sequences were closely related (>92% identity). Solovey and Primorye sequences shared 84% nucleotide identity with the prototype Hantaan 76-118. Phylogenetic analysis also indicated a close relationship between Solovey, Primorye, Amur, and other viruses identified in Russia, China, and Korea. Our findings suggest that the Korean field mouse (A. peninsulae) is the reservoir for a hantavirus that causes HFRS over a vast area of east Asia, including Far East Russia.

Animals↗

PubMed2002

Genetic and biological comparison of tick-borne encephalitis viruses from Hokkaido and far-eastern Russia.

We compared the biological properties of Oshima 5-10 (tick-borne encephalitis [TBE] virus isolated in Hokkaido, Japan) and Sofjin-HO (Far-Eastern subtype TBE virus) including plaque formation, virus replication and virus protein synthesis in BHK-21 cell cultures to reveal strain differences. We also determined the complete nucleotide sequences of both strains and compared the deduced amino acid sequences. Plaques of Oshima 5-10 were smaller than those of Sofjin-HO. Virus titers in culture fluid of Oshima 5-10 were 1/100 of those of Sofjin-HO at 9 and 12 hr after infection. Less viral protein and RNA syntheses of strain Oshima 5-10 was observed than with Sofjin-HO. Genetic analysis revealed 1.4% of amino acids to differ with Sofjin-HO. No difference between the two strains was detected in the motif sequence of the viral enzyme, cleavage sites of viral protein or glycosylation sites of NS1.

Amino Acid Sequence↗

PubMed2001

Distribution and characterization of tick-borne encephalitis viruses from Siberia and far-eastern Asia.

In this study, tick-borne encephalitis (TBE) viruses from Siberia and far-eastern Asia were characterized in order to determine virus subtype distribution. TBE viruses were isolated from ticks (Ixodes persulcatus) collected in the far-eastern (Khabarovsk and Vladivostok) and Siberian (Irkutsk) regions of Russia in 1999. Phylogenetic analysis showed that isolates formed distinct clusters of far-eastern and Siberian subtypes. There was also a minor difference in antigenicity between the Irkutsk isolates and other TBE virus strains, as demonstrated by the reactivity of monoclonal antibodies. Amino acid alignments of the E gene showed that the Irkutsk isolates had a single amino acid change at position 234 (Q or H); this amino acid position is considered to be a 'signature' of Siberian subtype TBE viruses. Strains isolated in Irkutsk also exhibited equivalent or somewhat higher virulence in mice compared with far-eastern TBE virus isolates. All viruses isolated in this study (i.e. far-east Asian and Siberian isolates) have 3' non-coding regions (NCRs) of almost the same length, which contrasts with the various sizes of 3'NCRs of other TBE viruses strains reported previously. The data presented in this study show that the 3'NCR is uniform among TBE viruses isolated from Siberia and far-eastern Asia and that the 3'NCR is essential for TBE virus growth in tick and/or rodent host cells.

3' Untranslated Regions↗

PubMed1999

Phylogenetic and virulence analysis of tick-borne encephalitis viruses from Japan and far-Eastern Russia.

We have previously reported that tick-borne encephalitis (TBE) is endemic in a specific area of Hokkaido, Japan. In Oshima, the southern part of Hokkaido, TBE virus was isolated from sentinel dogs, ticks and rodents in 1995 and 1996. To identify when these TBE viruses emerged in Hokkaido, the times of divergence of TBE virus strains isolated in Oshima and far-eastern Russia were estimated. TBE virus was isolated in Khabarovsk in 1998 and the nucleotide sequences of viral envelope protein genes of isolates from Oshima and Khabarovsk were compared. From the synonymous substitution rate of these virus strains, the lineage divergence time of these TBE virus strains was predicted phylogenetically to be about 260-430 years ago. Furthermore, the virulence of TBE virus isolates from Oshima and Khabarovsk were compared in a mouse model. The results showed that the isolates possessed very similar virulence in mice. This report provides evidence that the Oshima strains of TBE virus in Hokkaido emerged from far-eastern Russia a few hundred years ago and this explains why these strains possess virulence similar to the TBE viruses isolated in Russia.

Amino Acid Sequence↗