Kentaro Yamada

Improved Recovery of Rabies Virus from Cloned cDNA Using a Vaccinia Virus-Free Reverse Genetics System

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.

Rescue of Rabies Virus from Cloned cDNA and Identification of the Pathogenicity-Related Gene: Glycoprotein Gene Is Associated with Virulence for Adult Mice

ABSTRACT 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.

Multigenic Relation to the Attenuation of Rabies Virus

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.

Characterization of M gene-deficient rabies virus with advantages of effective immunization and safety as a vaccine strain

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‐HLAM, using a reverse genetics system of rabies virus RC‐HL strain to develop a novel type of vaccine. RC‐HLAM 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‐HLAM 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‐HLAM showed no symptoms. The neutralizing antibody against rabies virus was successfully induced by intramuscular immunization with 105 focus‐forming units of RC‐HLAM but not UV‐inactivated RC‐HL. Intranasal immunization with RC‐HLAM 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‐HLAM is a candidate for a novel rabies vaccine that is safer and more effective than are current vaccines.

Region at amino acids 164 to 303 of the rabies virus glycoprotein plays an important role in pathogenicity for adult mice

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.

Efficacy of Favipiravir (T-705) in Rabies Postexposure Prophylaxis

Rabies is a fatal encephalitis caused by rabies virus (RABV), and no antiviral drugs for RABV are currently available. We report for the first time the efficacy of favipiravir (T-705) against RABV in vitro and in vivo. T-705 produced a significant, 3–4 log10 reduction in the multiplication of street and fixed RABV strains in mouse neuroblastoma Neuro-2a cells, with half-maximal inhibitory concentrations of 32.4 µM and 44.3 µM, respectively. T-705 significantly improved morbidity and mortality among RABV-infected mice when orally administered at a dose of 300 mg/kg/day for 7 days, beginning 1 hour after inoculation. T-705 significantly reduced the rate of virus positivity in the brain. Furthermore, the effectiveness of T-705 was comparable to that of equine rabies virus immunoglobulin for postexposure prophylaxis. Collectively, our results suggest that T-705 is active against RABV and may serve as a potential alternative to rabies immunoglobulin in rabies postexposure prophylaxis.

Characterization of Recombinant Rabies Virus Carrying Double Glycoprotein Genes

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.

Amino acid at position 95 of the matrix protein is a cytopathic determinant of rabies virus

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 CPE 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 CPE 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.