Mutsuyo Takayama-Ito

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.

Rabies Virus-Induced Activation of Mitogen-Activated Protein Kinase and NF-κB Signaling Pathways Regulates Expression of CXC and CC Chemokine Ligands in Microglia

ABSTRACT Following virus infection of the central nervous system, microglia, the ontogenetic and functional equivalents of macrophages in somatic tissues, act as sources of chemokines, thereby recruiting peripheral leukocytes into the brain parenchyma. In the present study, we have systemically examined the growth characteristics of rabies virus (RV) in microglia and the activation of cellular signaling pathways leading to chemokine expression upon RV infection. In RV-inoculated microglia, the synthesis of the viral genome and the production of virus progenies were significantly impaired, while the expression of viral proteins was observed. Transcriptional analyses of the expression profiles of chemokine genes revealed that RV infection, but not exposure to inactivated virions, strongly induces the expression of CXC chemokine ligand 10 (CXCL10) and CC chemokine ligand 5 (CCL5) in microglia. RV infection triggered the activation of signaling pathways mediated by mitogen-activated protein kinases, including p38, extracellular signal-regulated kinases 1 and 2 (ERK1/2), and c-Jun N-terminal kinase, and nuclear factor κB (NF-κB). RV-induced expression of CXCL10 and CCL5 was achieved by the activation of p38 and NF-κB pathways. In contrast, the activation of ERK1/2 was found to down-regulate CCL5 expression in RV-infected microglia, despite the fact that it was involved in partial induction of CXCL10 expression. Furthermore, NF-κB signaling upon RV infection was augmented via a p38-mediated mechanism. Taken together, these results indicate that the strong induction of CXCL10 and CCL5 expression in microglia is precisely regulated by the activation of multiple signaling pathways through the recognition of RV infection.

Suppressive effect of simvastatin on interferon-β-induced expression of CC chemokine ligand 5 in microglia

Despite the pivotal role of microglia in immune system of the brain, a growing body of evidence suggests that the excessive microglial activation provokes neuronal and glial damages, leading to neurodegenerative and neuroinflammatory disorders. The 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors, or statins, have recently received much attention for their suppressive effects on inflammation in the central nervous system. In the current study, we have examined the statin-mediated inhibition of microglial function, especially that of chemokine production. Stimulation of microglial cells with interferon-beta (IFN-beta) resulted in the expression of CC chemokine ligand 5 (CCL5), a major chemoattractant of inflammatory cells. Microglial CCL5 response was synergistically potentiated by costimulation with IFN-beta and tumor necrosis factor-alpha (TNF-alpha). The simvastatin treatment significantly diminished the microglial CCL5 expression induced by IFN-beta alone or by IFN-beta/TNF-alpha combination. In the presence of simvastatin, the IFN-beta-induced activation of Janus kinase (Jak)-signal transducer and activator of transcription (STAT) pathway was attenuated, although this compound had little or no effect on the TNF-alpha-evoked activation of nuclear factor kappaB and c-Jun N-terminal kinase pathways. In addition, chemical inhibitor of Jak-STAT signaling significantly diminished the IFN-beta-induced expression of CCL5 in microglia. Taken together, these results suggest that simvastatin suppresses the IFN-beta-induced expression of CCL5 via down-regulation of Jak-STAT signaling pathway.

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.

Neonatal Herpes Encephalitis Caused by a Virologically Confirmed Acyclovir-Resistant Herpes Simplex Virus 1 Strain

ABSTRACT A neonate with herpes simplex virus 1 encephalitis was treated with intravenous acyclovir. During the course of therapy, the infection became intractable to the treatment and a mutation in the viral thymidine kinase gene (nucleotide G375T, amino acid Q125H) developed. This mutation was demonstrated in vitro to confer acyclovir resistance.

Progressive multifocal leukoencephalopathy developed in incomplete Heerfordt syndrome, a rare manifestation of sarcoidosis, without steroid therapy responding to cidofovir

Progressive multifocal leukoencephalopathy (PML) is a severe demyelinating disease of the central nervous system caused by the JC virus; the mortality rate is high and it is usually refractory to treatment. In non-HIV patients, PML occurs as a late consequence of hematologic malignancies or during prolonged immunosuppression for transplantation or autoimmune disease. We describe a 34-year-old PML patient with incomplete Heerfordt syndrome, a rare type of sarcoidosis, who had not received any immunosuppressants, including steroids, at the onset and who was clinically and radiologically responsive to the antiviral drug cidofovir.

Roles of NF-κB and MAPK signaling pathways in morphological and cytoskeletal responses of microglia to double-stranded RNA

Abstract Following virus infection of the central nervous system, microglia become activated and undergo morphological as well as functional transformations, thereby initiating effective antiviral actions. Herein, we have examined the contribution of nuclear factor κB (NF-κB) and mitogen-activated protein kinase (MAPK) signaling pathways to cell shape determination and cytoskeletal organization in microglia upon stimulation with double-stranded RNA (dsRNA), a conserved molecular pattern of virus infection. Under non-proliferative condition, microglial MG6-1 cells displayed a distinctive morphology with spinescent processes and small somata. Following dsRNA stimulation, the process-bearing microglial cells exhibited swift and drastic changes in cell morphology, filamentous actin (F-actin) structure, and intracellular signaling. In the dsRNA-stimulated microglial cells, the activation of c-Jun N-terminal kinase (JNK) pathway was involved in morphological alteration into an ameboid state. We also found that p38 signaling pathway negatively regulates the formation of cytoplasmic vacuoles in microglial cells. Furthermore, the dsRNA-induced accumulation of F-actin was partly mediated by NF-κB, JNK, and p38 pathways. These results indicate that NF-κB and MAPK signaling pathways mediate morphological and cytoskeletal changes during dsRNA-induced microglial activation.

Expression of herpes simplex virus type 1 recombinant thymidine kinase and its application to a rapid antiviral sensitivity assay.

Antiviral-resistant herpesvirus infection has become a great concern for immunocompromised patients. Herpes simplex virus type 1 (HSV-1) infections are treated with viral thymidine kinase (vTK)-associated drugs such as acyclovir (ACV), and most ACV-resistance (ACV(r)) is due to mutations in the vTK. The standard drug sensitivity test is usually carried out by the plaque reduction assay-based method, which requires over 10 days. To shorten the time required, a novel system was developed by the concept, in which 293T cells transiently expressing recombinant vTK derived from the test sample by transfection of the cells with an expression vector were infected with vTK-deficient and ACV(r) HSV-1 (TAR), and then cultured in a maintenance medium with or without designated concentrations of ACV, ganciclovir (GCV) and brivudine (BVdU). The replication of TAR was strongly inhibited by ACV, GCV and BVdU in 293T cells expressing recombinant vTK of the ACV-sensitive HSV-1, whereas replication was not or slightly inhibited in cells expressing the recombinant vTK of highly resistant or intermediately resistant HSV-1, respectively. An inverse correlation was demonstrated in the 50% effective concentrations (EC(50)s) and inhibitory effects of these compounds on the replication of TAR among ACV(s) and ACV(r) HSV-1 clones. These results indicate that the EC(50)s of the vTK-associated drugs including ACV can be assumed by measuring the inhibitory effect of drugs in 293T cells expressing recombinant vTK of the target virus. The newly developed antiviral sensitivity assay system for HSV-1 makes it possible to estimate EC(50) for vTK-associated drugs, when whole vTK gene is available for use by gene amplification directly from lesions samples or from virus isolates.