論文 - 山田 健太郎
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Determination of the 5'-terminal sequence of subgenomic RNA of hepatitis E virus strains in cultured cells
Koji Ichiyama, Kentaro Yamada, Toshinori Tanaka, Shigeo Nagashima, Jirintai, Masaharu Takahashi, Hiroaki Okamoto
ARCHIVES OF VIROLOGY 154 ( 12 ) 1945 - 1951 2009年12月
記述言語：日本語 掲載種別：研究論文（学術雑誌） 出版者・発行元：SPRINGER WIEN
Using RNA preparations extracted from PLC/PRF/5 cells transfected with infectious genotype 3 hepatitis E virus (HEV) cDNA clones or inoculated with a fecal suspension containing a genotype 4 HEV, the 5'-terminal sequence of a 2.2-kb subgenomic RNA of genotype 3 and 4 HEVs was determined. Despite an insertion of T after nucleotide 5116 or an ORF3-null mutation in genotype 4 HEV and/or one of the genotype 3 variants, it was found that the subgenomic RNA of genotype 3 and 4 HEVs initiates exclusively at nucleotide 5122 with the common sequence of 5'-GC, which is identical to that of the prototype genotype 1 HEV.
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Development and evaluation of a rapid neutralizing antibody test for rabies
Seiji Shiota, Kazuaki Mannen, Takashi Matsumoto, Kentaro Yamada, Takehito Yasui, Katsuyoshi Takayama, Yukuharu Kobayashi, Pakamatz Khawplod, Kazuyo Gotoh, Kamruddin Ahmed, Hidekatsu Iha, Akira Nishizono
JOURNAL OF VIROLOGICAL METHODS 161 ( 1 ) 58 - 62 2009年10月
記述言語：日本語 掲載種別：研究論文（学術雑誌） 出版者・発行元：ELSEVIER SCIENCE BV
The level of virus-neutralizing antibody, which plays a crucial role in the prevention of rabies, is determined by rabiesviruis (RABV) neutralizing test, which are time- and cost-consuming. In order to determine the level of neutralizing antibody in vaccinees, an easy and reliable method is needed. Based on the principle of immunochromatography, we developed a RAPINA (RAPId Neutralizing Antibody) test to determine the presence of neutralizing antibody in serum. In the RAPINA test, if neutralizing antibody equivalent to 0.5 IU/ml of serum sample are mixed with an optimal amount of inactivated RABV (iRABV) and are completely absorbed by the virus, none of the iRABV can bind with monoclonal antibody that recognizes the iRABV glycoprotein (G) on the test strip. A total of 115 human sera samples were tested. The sensitivity, specificity and accuracy of the RAPINA test compared with rapid fluorescent focus inhibition test (RFFIT) as a standard test, were 88.7, 91.9 and 90.4%, respectively. The RAPINA test is a simple, safe and rapid method, which can be a substitute for neutralizing tests that use live viruses, cultured cells and fluorescence microscopy. This test might be useful for screening a large number of sera. (C) 2009 Elsevier B.V. All rights reserved.
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Evaluation of a New Tumor Necrosis Factor-alpha-Inducing Membrane Protein of Helicobacter pylori as a Prophylactic Vaccine Antigen 査読あり
Kunimitsu Inoue, Seiji Shiota, Kentaro Yamada, Kazuyo Gotoh, Masami Suganuma, Toshio Fujioka, Kamruddin Ahmed, Hidekatsu Iha, Akira Nishizono
HELICOBACTER 14 ( 5 ) 135 - 143 2009年10月
記述言語：英語 掲載種別：研究論文（学術雑誌） 出版者・発行元：WILEY
Background:
Tumor necrosis factor (TNF)-alpha-inducing protein (Tip alpha) is a newly identified carcinogenic factor present in Helicobacter pylori. Tip alpha has the unique function of inducing TNF-alpha production by gastric cells in vitro and is assumed to be related with the development of gastritis and gastric cancer. We investigated the effects of vaccination with Tip alpha against H. pylori infection and analyzed the immune responses.
Methods:
C57BL/6 mice were immunized via the intranasal route with CpG, recombinant Tip alpha + CpG, and recombinant del-Tip alpha (a mutant of Tip alpha) + CpG. Eight weeks after the mice were infected with H. pylori (5 x 10<SU7</SU CFU), the number of colonizing bacteria in the stomach was calculated, and the histological severity of gastritis was evaluated. Levels of Tip alpha-specific IgG and IgA antibodies in mouse serum were measured by an enzyme-linked immunosorbent assay (ELISA). Local production of cytokines including Interleukin (IL)-10, TNF-alpha and Interferon (IFN)-gamma in gastric mucosa was also measured by real time-PCR.
Results:
Levels of Tip alpha-specific antibodies were significantly higher in Tip alpha-immunized and del-Tip alpha-immunized mice than in the infection control group. The numbers of colonizing bacteria were significantly reduced in Tip alpha-immunized mice (4.29 x 10<SU5</SU CFU/g) and del-Tip alpha immunized mice (2.5 x 10<SU5 </SUCFU/g) compared with infection control mice (5.7 x 10<SU6</SU CFU/g). The levels of IFN-gamma and IL-10 were significantly higher in del-Tip alpha-immunized mice than the infection control group.
Conclusion:
Vaccinations with Tip alpha and del-Tip alpha were effective against H. pylori infection. The inhibition of H. pylori colonization is associated mainly with Th1 cell-mediated immunity. -
ORF3 protein of hepatitis E virus is essential for virion release from infected cells 査読あり
Kentaro Yamada, Masaharu Takahashi, Yu Hoshino, Hideyuki Takahashi, Koji Ichiyama, Shigeo Nagashima, Toshinori Tanaka, Hiroaki Okamoto
JOURNAL OF GENERAL VIROLOGY 90 ( Pt 8 ) 1880 - 1891 2009年8月
担当区分：筆頭著者 記述言語：英語 掲載種別：研究論文（学術雑誌） 出版者・発行元：SOC GENERAL MICROBIOLOGY
The function of the hepatitis E virus (HEV) open reading frame 3 (ORF3) protein remains unclear. To elucidate the role of the ORF3 protein in the virus life cycle, an infectious cDNA clone (pJE03-1760F/wt) that can replicate efficiently in PLC/PRF/5 and A549 cells and release progeny into the culture medium was used to generate a derivative ORF3-deficient (Delta ORF3) mutant whose third in-frame AUG codon of ORF3 was mutated to GCA. The Delta ORF3 mutant in the culture medium of mutant RNA-transfected PLC/PRF/5 cells was able to infect and replicate within PLC/PRF/5 and A549 cells as efficiently as the wild-type pJE03-1760F/wt virus. However, less than 1/100 of the number of progeny was detectable in the culture medium of Delta ORF3 mutant-infected PLC/PRF/5 cells compared with wild-type-infected PLC/PRF/5 cells, and the HEV RNA level in the culture medium of Delta ORF3 mutant-infected A549 cells was below or near the limit of detection. An immunocapture PCR assay revealed that the ORF3 protein is present on the surface of cell-culture-generated wild-type HEV but not on the Delta ORF3 mutant. Wild-type HEV in the culture supernatant peaked at a sucrose density of 1.15-1.16 g ml(-1), in contrast with the Delta ORF3 mutant in culture supernatant, which banded at 1.27-1.28 g ml(-1), similar to HEV in cell lysate and faecal HEV. These results suggest that the ORF3 protein is responsible for virion egress from infected cells and is present on the surface of released HEV particles, which may be associated with lipids.
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Construction of an infectious cDNA clone of hepatitis E virus strain JE03-1760F that can propagate efficiently in cultured cells
Kentaro Yamada, Masaharu Takahashi, Yu Hoshino, Hideyuki Takahashi, Koji Ichiyama, Toshinori Tanaka, Hiroaki Okamoto
JOURNAL OF GENERAL VIROLOGY 90 ( Pt 2 ) 457 - 462 2009年2月
担当区分：筆頭著者 記述言語：英語 掲載種別：研究論文（学術雑誌） 出版者・発行元：SOC GENERAL MICROBIOLOGY
A full-length infectious cDNA clone (pJE03-1760F/wt) of a genotype 3 hepatitis E virus (HEV) (strain JE03-1760F) obtained from a faecal specimen was constructed ill this study. Upon transfection of the capped in vitro transcripts of pJE03-1760F/wt into PLC/PRF/5 cells, the viral RNA levels in the culture supernatant started to increase on day 6 post-transfection (p.t.) and reached 10(7) copies ml(-1) on day 28 p.t. Detection of increasing numbers of cells with ORF2 protein expression by immunofluorescence assay at 5, 7, 11 and 15 days p.t. indicated the spread of HEV infection in cell culture. When the cDNA-derived virus in culture supernatant was inoculated into PLC/PRF/5 or A549 cells, it grew as efficiently as the faeces-derived virus in both cells, reaching 10(6) copies ml(-1) at 30 days post-inoculation. Our reverse genetics system for HEV that is usable in a robust cell-culture system will be useful for elucidation of the mechanism of HEV replication and functional roles of HEV proteins.
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Amino acid at position 95 of the matrix protein is a cytopathic determinant of rabies virus
Tetsuo Mita, Kenta Shimizu, Naoto Ito, Kentaro Yamada, Yuki Ito, Makoto Sugiyama, Nobuyuki Minamoto
VIRUS RESEARCH 137 ( 1 ) 33 - 39 2008年10月
記述言語：日本語 掲載種別：研究論文（学術雑誌） 出版者・発行元：ELSEVIER SCIENCE BV
The molecular mechanism involved in cytopathogenicity of rabies virus has not been fully elucidated yet. A fixed rabies virus Nishigahara strain does not induce clear cytopathic effect (CPE) in mouse neuroblastoma (NA) cells, whereas Ni-CE strain, which was established after 100 passages of Nishigahara strain in chicken embryo fibroblast cells, induces CIPE that is characterized by rounding, shrinkage and detachment of the cells. In this study, to identify which viral gene is associated with the CPE of Ni-CE Strain, we analyzed chimeric viruses between Nishigahara and Ni-CE strains generated by reverse genetics systems of both strains. We showed that the matrix gene of Ni-CE strain is responsible for the CPE in NA cells. It was also demonstrated by infection of Nishigahara and Ni-CE mutants with a single amino acid substitution in the matrix protein (M) that an amino acid at position 95 of M is a cytopathic determinant of the virus. We also demonstrated that the CPF is, at least partly, due to apoptosis. This is the first report of identification of an amino acid residue in a rabies virus protein that is important for the cytopathogenicity of the virus. (C) 2008 Elsevier B.V. All rights reserved.
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Mutational events during the primary propagation and consecutive passages of hepatitis E virus strain JE03-1760F in cell culture
Felipe R. Lorenzo, Toshinori Tanaka, Hideyuki Takahashi, Koji Ichiyama, Yu Hoshino, Kentaro Yamada, Jun Inoue, Masaharu Takahashi, Hiroaki Okamoto
VIRUS RESEARCH 137 ( 1 ) 86 - 96 2008年10月
記述言語：日本語 掲載種別：研究論文（学術雑誌） 出版者・発行元：ELSEVIER SCIENCE BV
We recently developed a cell culture system for hepatitis E virus (HEV) in PLC/PRF/5 cells, using a genotype 3 HEV (JE03-1760F strain). Thirteen generations of consecutive passages of culture supernatant were successfully carried out in PLC/PRF/5 cells, with the highest HEV load reaching 10(8) copies/ml in the culture medium. Based on continuous release of progenies into culture medium, 50% tissue culture infectivity doses were estimated to be 2.0 x 10(3) Copies for wild-type JE03-1760F and 1.4 x 10(2) copies for p 13 (progeny in the thirteenth passage). Earlier appearance and greater increase in the yield of progenies in the culture supernatant were evident in p13 compared with wild-type. The cell culture-produced variants in primary propagation (p0)and consecutive passages (p5 [fifth passage], p10 [tenth], and p13)differed from the wildtype virus by 1. 9, 18, and 19 nucleotides (nt), respectively, over the entire genome of 7226 nt, excluding the poly(A) tail. Three of five non-synonymous Mutations in p13 were shared by a variant (fifth passage) in another series of passages of JE03-1760F. These results suggest that adaptation of HEV variants to growth in vitro is associated with a limited number of mutations similar to hepatitis A virus. (C) 2008 Elsevier B.V. All Fights reserved.
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Monoclonal antibodies raised against the ORF3 protein of hepatitis E virus (HEV) can capture HEV particles in culture supernatant and serum but not those in feces 査読あり
Masaharu Takahashi, Kentaro Yamada, Yu Hoshino, Hideyuki Takahashi, Koji Ichiyama, Toshinori Tanaka, Hiroaki Okamoto
ARCHIVES OF VIROLOGY 153 ( 9 ) 1703 - 1713 2008年9月
記述言語：英語 掲載種別：研究論文（学術雑誌） 出版者・発行元：SPRINGER WIEN
Ten murine monoclonal antibodies (MAbs) against a synthetic peptide corresponding to the well-conserved, C-terminal 24-amino acid portion of ORF3 protein of hepatitis E virus (HEV) were produced and characterized. Immunofluorescent assays using the anti-ORF3 MAbs revealed accumulation of ORF3 protein in the cytoplasm of PLC/PRF/5 cells transfected with ORF3-expressing plasmid or inoculated with cell-culture-generated HEV. The anti-ORF3 MAbs could capture HEV particles in culture medium and serum at variable efficiency of up to 61 and 49%, respectively, but not those in feces. By sandwiching between immobilized and enzyme-labeled anti-ORF3 MAbs in ELISA, ORF3 antigen was detected in the culture media with an HEV RNA titer of > 10(6) copies/ml and increased in parallel with the increase in HEV load. HEV progenies in the culture supernatant, with ORF3 protein on the surface, banded at a low buoyant density of 1.15 g/cm(3) in sucrose. A representative anti-ORF3 MAb (TA0536) could partially neutralize the infection of cell-culture-generated HEV in a cell culture system. These results indicate that ORF3 protein, at least its C-terminal portion, is present on the surface of HEV virions released from infected cells and support a previously proposed assumption that ORF3 protein is associated with virus release from infected cells.
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Adult measles 招待あり
Yamada K., Okada H.
Nippon rinsho. Japanese journal of clinical medicine 65 Suppl 3 364 - 367 2007年3月
担当区分：筆頭著者 記述言語：日本語 掲載種別：研究論文（学術雑誌） 出版者・発行元：Nippon rinsho. Japanese journal of clinical medicine
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Involvement of nucleoprotein, phosphoprotein, and matrix protein genes of rabies virus in virulence for adult mice
Kenta Shimizu, Naoto Ito, Tetsuo Mita, Kentaro Yamada, Junji Hosokawa-Muto, Makoto Sugiyama, Nobuyuki Minamoto
VIRUS RESEARCH 123 ( 2 ) 154 - 160 2007年2月
記述言語：日本語 掲載種別：研究論文（学術雑誌） 出版者・発行元：ELSEVIER SCIENCE BV
Rabies virus Ni-CE strain causes nonlethal infection in adult mice after intracerebral inoculation, whereas the parental Nishigahara strain kills mice. In this study, to identify viral gene(s) related to the difference in pathogenicity between Ni-CE and Nishigahara strains, we generated chimeric viruses with respective genes of the virulent Nishigahara strain in the background of the avirudent Ni-CE genome. Since chimeric viruses, which had the N, P, or M genes of the Nishigahara strain, respectively, killed adult mice after intracerebral inoculation, it became evident that the N, P, and M genes are related to the difference in pathogenicity between Ni-CE and Nishigahara strains. Previously, we showed that the G gene is a major contributor to the difference in pathogenicity between another avirulent strain, RC-HL, and the parental Nishigahara strain. These results imply that the attenuation mechanism of the Ni-CE strain is different from that of the RC-HL strain, thus suggesting that rabies virus can be attenuated by diverse mechanisms. This is the first report of changes in viral genes other than the G gene of rabies virus causing the reversion of pathogenicity of an avirulent strain. (c) 2006 Elsevier B.V. All rights reserved.
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Multiple amino acids in the glycoprotein of rabies virus are responsible for pathogenicity in adult mice
M Takayama-Ito, N Ito, K Yamada, M Sugiyama, N Minamoto
VIRUS RESEARCH 115 ( 2 ) 169 - 175 2006年2月
記述言語：日本語 掲載種別：研究論文（学術雑誌） 出版者・発行元：ELSEVIER SCIENCE BV
We have reported that the region between amino acids 164 and 303 in the glycoprotein of rabies Nishigahara strain is important for lethality in adult mice. The region contains nine amino acid substitutions between the virulent Nishigahara and the avirulent RC-HL strains. In order to determine key residues for the pathogenicity, two chimeric strains and seven mutants were generated and examined for pathogenicities. The R(G 242/255/268) strain, in which amino acids at positions 242, 255, and 268 were replaced by those from the Nishigahara strain in the genomic backbone of the RC-HL strain, showed the same lethality as that of the Nishigahara strain in mice. Mutants in which one or two of these three amino acids were replaced by those from the Nishigahara strain did not revert to the lethality of the R(G 242/255/268) strain. These results demonstrate that at least these three amino acids are related to enhancement of pathogenicity. It is thought that multiple amino acids of the G protein are related to the pathogenicity of rabies viruses. (c) 2005 Elsevier B.V. All rights reserved.
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Characterization of recombinant rabies virus carrying double glycoprotein genes 査読あり
J Hosokawa-Muto, N Ito, K Yamada, K Shimizu, M Sugiyama, N Minamoto
MICROBIOLOGY AND IMMUNOLOGY 50 ( 3 ) 187 - 196 2006年
記述言語：英語 掲載種別：研究論文（学術雑誌） 出版者・発行元：CENTER ACADEMIC PUBL JAPAN
A recombinant rabies virus carrying double glycoprotein (G) genes, R(NPMGGL) strain, was generated by a reverse genetics system utilizing cloned cDNA of the RC-HL strain, and the biological properties of the virus were compared to those of the recombinant RC-HL (rRC-HL) strain. The extents of virus growth in cultured cells and virulence for adult mice of the R(NPMGGL) strain were almost same as those of the rRC-HL strain, while G protein content of the purified R(NPMGGL) virion and G protein expression level in R(NPMGGL)-infected cells were 1.5-fold higher than those of the rRC-HL strain. As a result of serial passages of the R(NPMGGL) strain in cultured cells, the expression level of G protein in cultured cells infected with the passaged R(NPMGGL) strain was maintained and virus titers rose with adaptation to the cultured cells. Furthermore, analysis of neutralization titers in mice immunized with UV-inactivated virus suggested that the R(NPMGGL) strain had higher immunogenicity than that of the rRC-HL strain. The results suggest that the R(NPMGGL) strain carrying double G genes might be a useful candidate for development of a new inactivated rabies vaccine.
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Multigenic relation to the attenuation of rabies virus 査読あり
K Yamada, N Ito, M Takayama-Ito, M Sugiyama, N Minamoto
MICROBIOLOGY AND IMMUNOLOGY 50 ( 1 ) 25 - 32 2006年
担当区分：筆頭著者 記述言語：英語 掲載種別：研究論文（学術雑誌） 出版者・発行元：CENTER ACADEMIC PUBL JAPAN
Rabies virus Nishigahara strain causes lethal infection in adult mice after intracerebral inoculation. On the other hand, the RC-HL strain, derived from the Nishigahara strain, does not cause lethal infection in adult mice. We previously demonstrated that a chimeric virus, R(G), with the open reading frame of the G gene (G-ORF) from the Nishigahara strain in the background of the RC-HL genome, is virulent. Reversely, in order to demonstrate that the G gene of the RC-HL strain is related to the attenuated phenotype, we established a reverse genetics system of the Nishigahara strain and generated a chimeric virus, Ni(G), with the G-ORF from RC-HL in the background of the Nishigahara genome. Contrary to our prediction, Ni(G) killed adult mice after intracerebral inoculation with neuropathic symptoms like those of Nishigahara strain infection. Therefore, the G-ORF of the RC-HL strain is not the sole determinant of the attenuated phenotype. In additional investigation, we examined other genes, including N, P, M and L genes, and generated chimeric viruses exhaustively. We found that chimeric viruses with a single gene from the RC-HL were not attenuated and that chimeric viruses with the G-ORF and at least one other ORF from the RC-HL were attenuated. In conclusion, attenuation from the Nishigahara to RC-HL strain is multigenic.
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Ito N., Sugiyama M., Yamada K., Shimizu K., Takayama-Ito M., Hosokawa J., Minamoto N.
Microbiology and Immunology 49 ( 11 ) 971 - 979 2005年
記述言語：英語 掲載種別：研究論文（学術雑誌） 出版者・発行元：Microbiology and Immunology
Matrix (M) protein of rabies virus is known to play an important role in assembly and budding of the progeny virus. We generated an M gene-deficient rabies virus, RC-HLΔM, using a reverse genetics system of rabies virus RC-HL strain to develop a novel type of vaccine. RC-HLΔM infection was confined within a single cell in mouse neuroblastoma cells. This deficient virus failed to generate the progeny virus in the cells. In contrast, RC-HLΔM propagated in BHK cells inductively expressing M protein. Suckling and adult mice inoculated intracerebrally with the parental RC-HL strain showed lethal infection and transient body weight loss, respectively, whereas both suckling and adult mice inoculated with RC-HLΔM showed no symptoms. The neutralizing antibody against rabies virus was successfully induced by intramuscular immunization with 10 focus-forming units of RC-HLΔM but not UV-inactivated RC-HL. Intranasal immunization with RC-HLΔM resulted in almost the same antibody titer to rabies virus as that in the case of immunization with live RC-HL strain. These findings indicate that RC-HLΔM is a candidate for a novel rabies vaccine that is safer and more effective than are current vaccines. 5
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Characterization of M gene-deficient rabies virus with advantages of effective immunization and safety as a vaccine strain. 査読あり
Ito N, Sugiyama M, Yamada K, Shimizu K, Takayama-Ito M, Hosokawa J, Minamoto N
Microbiology and immunology 49 ( 11 ) 971 - 979 2005年
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Region at amino acids 164 to 303 of the rabies virus glycoprotein plays an important role in pathogenicity for adult mice
M Takayama-Ito, N Ito, K Yamada, N Minamoto, M Sugiyama
JOURNAL OF NEUROVIROLOGY 10 ( 2 ) 131 - 135 2004年4月
記述言語：日本語 掲載種別：研究論文（学術雑誌） 出版者・発行元：TAYLOR & FRANCIS INC
The authors have previously reported that the glycoprotein of the pathogenic Nishigahara strain of rabies virus is required to lethality for adult mice. A cluster region of amino acid substitutions exists at the positions 164 to 303 on the glycoprotein between avirulent and virulent strains. In this study, the authors generated a chimeric strain having the region at the positions 164 to 303 of the glycoprotein derived from the pathogenic Nishigahara strain in the genetic background of the avirulent RC-HL strain. The chimeric R(G 164-303) strain restores the lethality for adult mice. This result clearly shows that the region at the position 164 to 303 of glycoprotein plays an important role in the lethality for adult mice. Moreover, the authors observed that the lethality for adult mice correlated well with the viral growth in a brain but not with the pH-dependent fusion activity in vitro.
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Ito N., Takayama-Ito M., Yamada K., Hosokawa J., Sugiyama M., Minamoto N.
Microbiology and Immunology 47 ( 8 ) 613 - 617 2003年
記述言語：日本語 掲載種別：研究論文（学術雑誌） 出版者・発行元：Microbiology and Immunology
To improve efficiency of recovery of rabies virus from cloned cDNA, we established a BHK cell clone that stably expresses T7 RNA polymerase, which we named BHK/T7-9. We also constructed new helper plasmids for expression of nucleoprotein and RNA polymerase of the RC-HL strain using the pTM1 plasmid vector, which makes the T7 RNA polymerase-transcripts from the plasmid cap-independent for translation. After co-transfection of these helper plasmids and the previously constructed full-length genome plasmid of the RC-HL strain to BHK/T7-9 cells, recombinant rabies virus was efficiently recovered from the cloned cDNA.
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Improved recovery of rabies virus from cloned cDNA using a vaccinia virus-free reverse genetics system 査読あり
N Ito, M Takayama-Ito, K Yamada, J Hosokawa, M Sugiyama, N Minamoto
MICROBIOLOGY AND IMMUNOLOGY 47 ( 8 ) 613 - 617 2003年
記述言語：英語 掲載種別：研究論文（学術雑誌） 出版者・発行元：CENTER ACADEMIC PUBL JAPAN
To improve efficiency of recovery of rabies virus from cloned cDNA, we established a BHK cell clone that stably expresses T7 RNA polymerase, which we named BHK/T7-9. We also constructed new helper plasmids for expression of nucleoprotein and RNA polymerase of the RC-HL strain using the pTM1 plasmid vector, which makes the T7 RNA polymerase-transcripts from the plasmid cap-independent for translation. After co-transfection of these helper plasmids and the previously constructed full-length genome plasmid of the RC-HL strain to BHK/T7-9 cells, recombinant rabies virus was efficiently recovered from the cloned cDNA.
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Rescue of rabies virus from cloned cDNA and identification of the pathogenicity-related gene: Glycoprotein gene is associated with virulence for adult mice 査読あり
N Ito, M Takayama, K Yamada, M Sugiyama, N Minamoto
JOURNAL OF VIROLOGY 75 ( 19 ) 9121 - 9128 2001年10月
記述言語：英語 掲載種別：研究論文（学術雑誌） 出版者・発行元：AMER SOC MICROBIOLOGY
In order to identify the viral gene related to the pathogenicity of rabies virus, we tried to establish a reverse genetics system of the attenuated RC-HL strain, which causes nonlethal infection in adult mice after intracerebral inoculation. A full-length genome plasmid encoding the complete antigenomic cDNA of the RC-HL strain and helper plasmids containing cDNAs of the complete open reading frame of the N, P, and L genes, respectively, were constructed. After transfection of these plasmids into BHK-21 cells infected with the T7 RNA polymerase-expressing vaccinia virus, infectious rabies virus with almost the same biological properties as those of the wild-type RC-HL strain was rescued. Using this reverse genetics system of the RC-HL strain, we generated a chimeric virus with the open reading frame of the glycoprotein gene from the parent Nishigahara strain, which kills adult mice after intracerebral inoculation, in the background of the RC-HL genome. Since the chimeric virus killed adult mice following intracerebral inoculation, it became evident that the open reading frame of the glycoprotein gene is related to the pathogenicity of the Nishigahara strain for adult mice.