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Gene therapy (GT) products created using adeno-associated virus (AAV) vectors tend to exhibit toxicity via immune reactions, but other mechanisms of toxicity remain incompletely understood. We examined the cardiotoxicity of an overexpressed transgenic protein. Male C57BL/6J mice were treated with a single intravenous dose of product X, an AAV-based GT product, at 2.6 × 10 13 vg/kg. Necropsies were performed at 24 h, 7 days, and 14 days after dosing. Pathological examination and gene expression analysis were performed on the heart. Histopathologically, hypertrophy and vacuolar degeneration of cardiomyocytes and fibrosis were observed 14 days after dosing. Immunohistochemistry for endoplasmic reticulum (ER) stress-related proteins revealed increased positive reactions for glucose-regulated protein 78 and C/EBPR homologous protein in cardiomyocytes 7 days after dosing, without histopathological abnormalities. Fourteen days after dosing, some cardiomyocytes showed positivity for PKR-like endoplasmic reticulum kinase and activating transcription factor 4 expression. Ultrastructurally, increases in the ER and cytosol were observed in cardiomyocytes 7 days after dosing, along with an increase in the number of Golgi apparatus compartments 14 days after dosing. The tissue concentration of the transgene product protein increased 7 days after dosing. Gene expression analysis showed upregulation of ER stress-related genes 7 days after dosing, suggesting activation of the PKR-like ER kinase pathway of the unfolded protein reaction (UPR). Thus, the cardiotoxicity induced by product X was considered to involve cell damage caused by the overexpression of the product protein accompanied by UPR. Marked UPR activation may also cause toxicity of AAV-based GT products., Competing Interests: Kyohei Yasuno, Ryo Watanabe, Rumiko Ishida, Hirofumi Kondo, Masako Imaoka, and Yoshimi Tsuchiya are employees of Daiichi Sankyo Co., Ltd. Keiko Okado is an employee of Daiichi Sankyo RD Novare Co., Ltd., (©2024 The Japanese Society of Toxicologic Pathology.)

Tick-borne encephalitis virus (TBEV) causes a severe and potentially fatal neuroinfection in humans. Despite its high medical relevance, no specific antiviral therapy is currently available. Here we demonstrate that treatment with a nucleoside analog, 7-deaza-2'- C -methyladenosine (7-deaza-2'-CMA), substantially improved disease outcomes, increased survival, and reduced signs of neuroinfection and viral titers in the brains of mice infected with a lethal dose of TBEV. To investigate the mechanism of action of 7-deaza-2'-CMA, two drug-resistant TBEV clones were generated and characterized. The two clones shared a signature amino acid substitution, S603T, in the viral NS5 RNA-dependent RNA polymerase (RdRp) domain. This mutation conferred resistance to various 2'- C -methylated nucleoside derivatives, but no cross-resistance was seen with other nucleoside analogs, such as 4'- C -azidocytidine and 2'-deoxy-2'-beta-hydroxy-4'-azidocytidine (RO-9187). All-atom molecular dynamics simulations revealed that the S603T RdRp mutant repels a water molecule that coordinates the position of a metal ion cofactor as 2'- C -methylated nucleoside analogs approach the active site. To investigate its phenotype, the S603T mutation was introduced into a recombinant TBEV strain (Oshima-IC) generated from an infectious cDNA clone and into a TBEV replicon that expresses a reporter luciferase gene (Oshima-REP-luc2A). The mutants were replication impaired, showing reduced growth and a small plaque size in mammalian cell culture and reduced levels of neuroinvasiveness and neurovirulence in rodent models. These results indicate that TBEV resistance to 2'- C -methylated nucleoside inhibitors is conferred by a single conservative mutation that causes a subtle atomic effect within the active site of the viral NS5 RdRp and is associated with strong attenuation of the virus. IMPORTANCE This study found that the nucleoside analog 7-deaza-2'- C -methyladenosine (7-deaza-2'-CMA) has high antiviral activity against tick-borne encephalitis virus (TBEV), a pathogen that causes severe human neuroinfections in large areas of Europe and Asia and for which there is currently no specific therapy. Treating mice infected with a lethal dose of TBEV with 7-deaza-2'-CMA resulted in significantly higher survival rates and reduced the severity of neurological signs of the disease. Thus, this compound shows promise for further development as an anti-TBEV drug. It is important to generate drug-resistant mutants to understand how the drug works and to develop guidelines for patient treatment. We generated TBEV mutants that were resistant not only to 7-deaza-2'-CMA but also to a broad range of other 2'- C -methylated antiviral medications. Our findings suggest that combination therapy may be used to improve treatment and reduce the emergence of drug-resistant viruses during nucleoside analog therapy for TBEV infection., (Copyright © 2017 American Society for Microbiology.)

Neurological diseases caused by encephalitic flaviviruses are severe and associated with high levels of mortality. However, little is known about the detailed mechanisms of viral replication and pathogenicity in the brain. Previously, we reported that the genomic RNA of tick-borne encephalitis virus (TBEV), a member of the genus Flavivirus , is transported and replicated in the dendrites of neurons. In the present study, we analyzed the transport mechanism of the viral genome to dendrites. We identified specific sequences of the 5' untranslated region of TBEV genomic RNA that act as a cis -acting element for RNA transport. Mutated TBEV with impaired RNA transport in dendrites caused a reduction in neurological symptoms in infected mice. We show that neuronal granules, which regulate the transport and local translation of dendritic mRNAs, are involved in TBEV genomic RNA transport. TBEV genomic RNA bound an RNA-binding protein of neuronal granules and disturbed the transport of dendritic mRNAs. These results demonstrated a neuropathogenic virus hijacking the neuronal granule system for the transport of viral genomic RNA in dendrites, resulting in severe neurological disease., Competing Interests: The authors declare no conflict of interest.

Bio-inspired surfaces that exhibit high contact angle and low contact angle hysteresis for various liquids and demonstrate mechanical durability and anti-smudge properties are of interest for various applications. The fabrication of such surfaces has often involved complex or expensive processes, required techniques that may not be suitable for various substrates and particles, may require surface post-treatment, or may lack durability. Dual layered coatings of roughness-induced superomniphobic surfaces that demonstrate good mechanical durability were fabricated on glass substrates using hydrophobic SiO2 nanoparticles and low surface energy fluorobinders using dip coating and spray coating techniques. The particle-to-binder ratio was optimized for contact angles of interest. The mechanical durability of these coatings was examined under mechanical rubbing action. The anti-smudge properties were examined by wiping an artificially contaminated coating using oil-impregnated microfiber cloth., (Copyright © 2013 Elsevier Inc. All rights reserved.)

The formation and proliferation of the dikaryon in the agaricomycete Coprinopsis cinerea is controlled by the mating type genes, A and B. The B genes, which encode pheromones and pheromone receptors, control nuclear migration for dikaryosis as well as the fusion of the clamp cell with the subterminal cell while the A genes, which encode two classes of homeodomain proteins, control conjugate nuclear division associated with clamp connection development. We characterized the mutant, B28, which was newly isolated as a strain that fails to form a primary hyphal knot, the first visible sign toward fruiting, from a homokaryotic fruiting strain after REMI mutagenesis. Detailed phenotypic analysis revealed that strain B28 exhibits, in addition to the fruiting defect, a defect in A-regulated clamp cell morphogenesis as well as a defect in B-regulated nuclear migration for dikaryosis. The mutant clamp cells are unique in that they continue growing like branches without fusing with the subterminal cells, in contrast to the unfused pseudoclamp which are normally formed in A-on B-off strains, providing evidence for the existence of an as yet unidentified mechanism for the growth suppression of the clamp cell. Molecular analysis revealed that the gene responsible for the phenotypes, designated Cc.ubc2, encodes a protein similar to Ubc2, an adaptor protein for filamentous growth, pheromone response and virulence in the smut fungus Ustilago maydis. In addition, western blot analysis demonstrated that the Cc.ubc2-1 mutation blocks phosphorylation of a presumptive MAP kinase., (Copyright © 2011 Elsevier Inc. All rights reserved.)