Biology subjects

Kotaki, T.

Publications and source records attributed to Kotaki, T..

5 recordsLinked to original sources

bioRxiv2023

Construction of Fosmid-based SARS-CoV-2 replicons for antiviral drug screening and replication analyses in biosafety level 2 facilities

The coronavirus disease 2019 (COVID-19) pandemic, caused by severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), has necessitated the global development of countermeasures since its outbreak. However, current therapeutics and vaccines to stop the pandemic are insufficient and this is mainly because of the emergence of resistant variants, which requires the urgent development of new countermeasures, such as antiviral drugs. Replicons, self-replicating RNAs that do not produce virions, are a promising system for this purpose because they safely recreate viral replication, enabling antiviral screening in biosafety level (BSL)-2 facilities. We herein constructed three pCC2Fos-based RNA replicons lacking some open reading frames (ORF) of SARS-CoV-2: the {Delta}orf2-8, {Delta}orf2.4, and {Delta}orf2 replicons, and validated their replication in Huh-7 cells. The functionalities of the {Delta}orf2-8 and {Delta}orf2.4 replicons for antiviral drug screening were also confirmed. We conducted puromycin selection following the construction of the {Delta}orf2.4-puro replicon by inserting a puromycin-resistant gene into the {Delta}orf2.4 replicon. We observed the more sustained replication of the {Delta}orf2.4-puro replicon by puromycin pressure. The present results will contribute to the establishment of a safe and useful replicon system for analyzing SARS-CoV-2 replication mechanisms as well as the development of novel antiviral drugs in BSL-2 facilities.

microbiology↗

bioRxiv2021

Inactivation of SARS-CoV-2 and influenza A virus by spraying hypochlorous acid solution and hydrogen peroxide solution in the form of Dry Fog

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of coronavirus disease 2019 (COVID-19), is transmitted by droplet and contact infection. SARS-CoV-2 that adheres to environmental surfaces remains infectious for several days. We herein attempted to inactivate SARS-CoV-2 and influenza A virus adhering to an environmental surface by spraying aerosolized hypochlorous acid solution and hydrogen peroxide solution in the form of Dry Fog (fog that does not wet objects even if touched). SARS-CoV-2 and influenza virus were dried on plastic plates and placed into a test chamber for inactivation by the Dry Fog spraying of disinfectants. The results obtained showed that Dry Fog spraying inactivated SARS-CoV-2 and influenza A virus in time- and exposed disinfectant amount-dependent manners. SARS-CoV-2 was more resistant to the virucidal effects of aerosolized hypochlorous acid solution and hydrogen peroxide solution than influenza A virus; therefore, higher concentrations of spray solutions were required to inactivate SARS-CoV-2 than influenza A virus. The present results provide important information for the development of a strategy that inactivates SARS-CoV-2 and influenza A virus on environmental surfaces by spatial spraying.

microbiology↗

bioRxiv2020

An affinity-matured human monoclonal antibody targeting fusion loop epitope of dengue virus with in vivo therapeutic potency

Dengue virus (DENV), from the genus flavivirus of the family flaviviridae, causes serious health problems globally. Human monoclonal antibodies (HuMAb) can be used to elucidate the mechanisms of neutralization and antibody-dependent enhancement (ADE) of DENV infections, leading to the development of a vaccine or therapeutic antibodies. Here, we generated eight HuMAb clones from an Indonesian patient infected with DENV. These HuMAbs exhibited the typical characteristics of weak neutralizing antibodies including high cross-reactivity with other flaviviruses and targeting of the fusion loop epitope (FLE). However, one of the HuMAbs, 3G9, exhibited strong neutralization ability (NT50 < 0.1 {micro}g/ml) and possessed a high somatic hyper-mutation rate of the variable region, indicating affinity-maturation. Administration of this antibody significantly improved the survival rate of interferon-/{beta}/{gamma} receptor knockout C57BL/6 mice after a lethal DENV challenge. Additionally, Fc-modified 3G9 molecules that had lost their in vitro ADE activity showed significantly enhanced therapeutic potency in vivo and competed strongly with an ADE-prone antibody in vitro. Taken together, the affinity-matured FLE-targeting antibody 3G9 exhibits several promising features for therapeutic application including a low NT50 value, potential for pan-flavivirus infection treatment, and suppression of ADE. This study demonstrates the therapeutic potency of affinity-matured FLE-targeting antibodies.

microbiology↗

bioRxiv2020

A potent neutralizing mouse monoclonal antibody specific to dengue virus type 1 Mochizuki strain recognized a novel epitope around the N-67 glycan on the E protein: a possible explanation of dengue virus evolution regarding the acquisition of N-67 glycan

Analysis of the neutralizing epitope of dengue virus (DENV) is important for the development of an effective dengue vaccine. A potent neutralizing mouse monoclonal antibody named 7F4 was previously reported and, here, we further analyze the detailed epitope of this antibody. 7F4 recognized a novel conformational epitope close to the N-67 glycan on the E protein. This antibody was specific to the DENV that lacks N-67 glycan, including the Mochizuki strain. Interestingly, the Mochizuki strain acquired N-67 glycan by 7F4 selective pressure. DENVs might have evolved to escape from this antibody considering that most of currently circulating DENVs possess N-67 glycan. However, this suggests that 7F4 epitope might be useless as a vaccine target. Nevertheless, this study demonstrated the existence of epitopes competing for 7F4 epitope, which are involved in neutralization. This study describes the importance of antibodies recognizing epitopes near the N-67 glycan for future dengue vaccine development.

microbiology↗

bioRxiv2020

A PCR amplicon-based SARS-CoV-2 replicon for antiviral screening

The development of specific antiviral compounds to SARS-CoV-2 is an urgent task. One of the obstacles for the antiviral development is the requirement of biocontainment because infectious SARS-CoV-2 must be handled in a biosafety level-3 laboratory. Replicon, a non-infectious self-replicative viral RNA, could be a safe and effective tool for antiviral screening; however, SARS-CoV-2 replicon has not been reported yet. Herein, we generated a PCR-based SARS-CoV-2 replicon. Eight fragments covering the entire SARS-CoV-2 genome except S, E, and M genes were amplified with HiBiT-tag sequence by PCR. The amplicons were ligated and in vitro transcribed to RNA. The cells electroporated with the replicon RNA showed more than 3,000 times higher luminescence than MOCK control cells at 24 hours post-electroporation, indicating robust viral translation and RNA replication. The replication was drastically inhibited by remdesivir, an RNA polymerase inhibitor for SARS-CoV-2. The IC50 of remdesivir in this study was 0.29 M, generally consistent to the IC50 obtained using infectious SARS-CoV-2 in a previous study (0.77 M). Taken together, this system could be applied to the safe and effective antiviral screening without using infectious SARS-CoV-2. Because this is a transient replicon, further improvement including the establishment of stable cell line must be achieved.

microbiology↗