Evaluation of Broad Anti-Coronavirus Activity of Autophagy-Related Compounds Using Human Airway Organoids.

To deal with the broad spectrum of coronaviruses, including severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), that threaten human health, it is essential to not only drugs develop that target viral proteins but also consider drugs that target host proteins/cellular processes to protect them from being hijacked for viral infection and replication. To this end, it has been reported that autophagy is deeply involved in coronavirus infection. In this study, we used airway organoids to screen a chemical library of autophagic modulators to identify compounds that could potentially be used to fight against infections by a broad range of coronaviruses. Among the 80 autophagy-related compounds tested, cycloheximide and thapsigargin reduced SARS-CoV-2 infection efficiency in a dose-dependent manner. Cycloheximide treatment reduced the infection efficiency of not only six SARS-CoV-2 variants but also human coronavirus (HCoV)-229E and HCoV-OC43. Cycloheximide treatment also reversed viral infection-induced innate immune responses. However, even low-dose (1 µM) cycloheximide treatment altered the expression profile of ribosomal RNAs; thus, side effects such as inhibition of protein synthesis in host cells must be considered. These results suggest that cycloheximide has broad-spectrum anti-coronavirus activity in vitro and warrants further investigation.

Elucidation of the liver pathophysiology of COVID-19 patients using liver-on-a-chips.

SARS-CoV-2 induces severe organ damage not only in the lung but also in the liver, heart, kidney, and intestine. It is known that COVID-19 severity correlates with liver dysfunction, but few studies have investigated the liver pathophysiology in COVID-19 patients. Here, we elucidated liver pathophysiology in COVID-19 patients using organs-on-a-chip technology and clinical analyses. First, we developed liver-on-a-chip (LoC) which recapitulating hepatic functions around the intrahepatic bile duct and blood vessel. We found that hepatic dysfunctions, but not hepatobiliary diseases, were strongly induced by SARS-CoV-2 infection. Next, we evaluated the therapeutic effects of COVID-19 drugs to inhibit viral replication and recover hepatic dysfunctions, and found that the combination of anti-viral and immunosuppressive drugs (Remdesivir and Baricitinib) is effective to treat hepatic dysfunctions caused by SARS-CoV-2 infection. Finally, we analyzed the sera obtained from COVID-19 patients, and revealed that COVID-19 patients, who were positive for serum viral RNA, are likely to become severe and develop hepatic dysfunctions, as compared with COVID-19 patients who were negative for serum viral RNA. We succeeded in modeling the liver pathophysiology of COVID-19 patients using LoC technology and clinical samples.

Compact wide-field femtoliter-chamber imaging system for high-speed and accurate digital bioanalysis.

The femtoliter-chamber array is a bioanalytical platform that enables highly sensitive and quantitative analysis of biological reactions at the single-molecule level. This feature has been considered a key technology for "digital bioanalysis" in the biomedical field; however, its versatility is limited by the need for a large and expensive setup such as a fluorescence microscope, which requires a long time to acquire the entire image of a femtoliter-chamber array. To address these issues, we developed a compact and inexpensive wide-field imaging system (COWFISH) that can acquire fluorescence images with a large field of view (11.8 mm × 7.9 mm) and a high spatial resolution of ∼ 3 µm, enabling high-speed analysis of sub-million femtoliter chambers in 20 s. Using COWFISH, we demonstrated a CRISPR-Cas13a-based digital detection of viral RNA of SARS-CoV-2 with an equivalent detection sensitivity (limit of detection: 480 aM) and a 10-fold reduction in total imaging time, as compared to confocal fluorescence microscopy. In addition, we demonstrated the feasibility of COWFISH to discriminate between SARS-CoV-2-positive and -negative clinical specimens with 95% accuracy, showing its application in COVID-19 diagnosis. Therefore, COWFISH can serve as a compact and inexpensive imaging system for high-speed and accurate digital bioanalysis, paving a way for various biomedical applications, such as diagnosis of viral infections.

Structure of SARS-CoV-2 membrane protein essential for virus assembly.

The coronavirus membrane protein (M) is the most abundant viral structural protein and plays a central role in virus assembly and morphogenesis. However, the process of M protein-driven virus assembly are largely unknown. Here, we report the cryo-electron microscopy structure of the SARS-CoV-2 M protein in two different conformations. M protein forms a mushroom-shaped dimer, composed of two transmembrane domain-swapped three-helix bundles and two intravirion domains. M protein further assembles into higher-order oligomers. A highly conserved hinge region is key for conformational changes. The M protein dimer is unexpectedly similar to SARS-CoV-2 ORF3a, a viral ion channel. Moreover, the interaction analyses of M protein with nucleocapsid protein (N) and RNA suggest that the M protein mediates the concerted recruitment of these components through the positively charged intravirion domain. Our data shed light on the M protein-driven virus assembly mechanism and provide a structural basis for therapeutic intervention targeting M protein.

SARS-CoV-2 disrupts respiratory vascular barriers by suppressing Claudin-5 expression.

In the initial process of coronavirus disease 2019 (COVID-19), severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infects respiratory epithelial cells and then transfers to other organs the blood vessels. It is believed that SARS-CoV-2 can pass the vascular wall by altering the endothelial barrier using an unknown mechanism. In this study, we investigated the effect of SARS-CoV-2 on the endothelial barrier using an airway-on-a-chip that mimics respiratory organs and found that SARS-CoV-2 produced from infected epithelial cells disrupts the barrier by decreasing Claudin-5 (CLDN5), a tight junction protein, and disrupting vascular endothelial cadherin-mediated adherens junctions. Consistently, the gene and protein expression levels of CLDN5 in the lungs of a patient with COVID-19 were decreased. CLDN5 overexpression or Fluvastatin treatment rescued the SARS-CoV-2-induced respiratory endothelial barrier disruption. We concluded that the down-regulation of CLDN5 expression is a pivotal mechanism for SARS-CoV-2-induced endothelial barrier disruption in respiratory organs and that inducing CLDN5 expression is a therapeutic strategy against COVID-19.

Cell response analysis in SARS-CoV-2 infected bronchial organoids.

The development of an in vitro cell model that can be used to study severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) research is expected. Here we conducted infection experiments in bronchial organoids (BO) and an BO-derived air-liquid interface model (BO-ALI) using 8 SARS-CoV-2 variants. The infection efficiency in BO-ALI was more than 1,000 times higher than that in BO. Among the bronchial epithelial cells, we found that ciliated cells were infected with the virus, but basal cells were not. Ciliated cells died 7 days after the viral infection, but basal cells survived after the viral infection and differentiated into ciliated cells. Fibroblast growth factor 10 signaling was essential for this differentiation. These results indicate that BO and BO-ALI may be used not only to evaluate the cell response to SARS-CoV-2 and coronavirus disease 2019 (COVID-19) therapeutic agents, but also for airway regeneration studies.

Automated amplification-free digital RNA detection platform for rapid and sensitive SARS-CoV-2 diagnosis.

In the ongoing COVID-19 pandemic, rapid and sensitive diagnosis of viral infection is a critical deterrent to the spread of SARS-CoV-2. To this end, we developed an automated amplification-free digital RNA detection platform using CRISPR-Cas13a and microchamber device (opn-SATORI), which automatically completes a detection process from sample mixing to RNA quantification in clinical specimens within ~9 min. Using the optimal Cas13a enzyme and magnetic beads technology, opn-SATORI detected SARS-CoV-2 genomic RNA with a LoD of < 6.5 aM (3.9 copies µL-1), comparable to RT-qPCR. Additionally, opn-SATORI discriminated between SARS-CoV-2 variants of concern, including alpha, delta, and omicron, with 98% accuracy. Thus, opn-SATORI can serve as a rapid and convenient diagnostic platform for identifying several types of viral infections.

Non-Woven Infection Prevention Fabrics Coated with Biobased Cranberry Extracts Inactivate Enveloped Viruses Such as SARS-CoV-2 and Multidrug-Resistant Bacteria.

The Coronavirus Disease (COVID-19) pandemic is demanding the rapid action of the authorities and scientific community in order to find new antimicrobial solutions that could inactivate the pathogen SARS-CoV-2 that causes this disease. Gram-positive bacteria contribute to severe pneumonia associated with COVID-19, and their resistance to antibiotics is exponentially increasing. In this regard, non-woven fabrics are currently used for the fabrication of infection prevention clothing such as face masks, caps, scrubs, shirts, trousers, disposable gowns, overalls, hoods, aprons and shoe covers as protective tools against viral and bacterial infections. However, these non-woven fabrics are made of materials that do not exhibit intrinsic antimicrobial activity. Thus, we have here developed non-woven fabrics with antimicrobial coatings of cranberry extracts capable of inactivating enveloped viruses such as SARS-CoV-2 and the bacteriophage phi 6 (about 99% of viral inactivation in 1 min of viral contact), and two multidrug-resistant bacteria: the methicillin-resistant Staphylococcus aureus and the methicillin-resistant Staphylococcus epidermidis. The morphology, thermal and mechanical properties of the produced filters were characterized by optical and electron microscopy, differential scanning calorimetry, thermogravimetry and dynamic mechanical thermal analysis. The non-toxicity of these advanced technologies was ensured using a Caenorhabditis elegans in vivo model. These results open up a new prevention path using natural and biodegradable compounds for the fabrication of infection prevention clothing in the current COVID-19 pandemic and microbial resistant era.

Antiviral Face Mask Functionalized with Solidified Hand Soap: Low-Cost Infection Prevention Clothing against Enveloped Viruses Such as SARS-CoV-2.

Infection prevention clothing is becoming an essential protective tool in the current pandemic, especially because now we know that severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) can easily infect humans in poorly ventilated indoor spaces. However, commercial infection prevention clothing is made of fabrics that are not capable of inactivating the virus. Therefore, viral infections of symptomatic and asymptomatic individuals wearing protective clothing such as masks can occur through aerosol transmission or by contact with the contaminated surfaces of the masks, which are suspected as an increasing source of highly infectious biological waste. Herein, we report an easy fabrication method of a novel antiviral non-woven fabric containing polymer filaments that were coated with solidified hand soap. This extra protective fabric is capable of inactivating enveloped viruses such as SARS-CoV-2 and phage Φ6 within 1 min of contact. In this study, this antiviral fabric was used to fabricate an antiviral face mask and did not show any cytotoxic effect in human keratinocyte HaCaT cells. Furthermore, this antiviral non-woven fabric could be used for the fabrication of other infection prevention clothing such as caps, scrubs, shirts, trousers, disposable gowns, overalls, hoods, aprons, and shoe covers. Therefore, this low-cost technology could provide a wide range of infection-protective tools to combat COVID-19 and future pandemics in developed and underdeveloped countries.

Antimicrobial Face Shield: Next Generation of Facial Protective Equipment against SARS-CoV-2 and Multidrug-Resistant Bacteria.

Transparent materials used for facial protection equipment provide protection against microbial infections caused by viruses and bacteria, including multidrug-resistant strains. However, transparent materials used for this type of application are made of materials that do not possess antimicrobial activity. They just avoid direct contact between the person and the biological agent. Therefore, healthy people can become infected through contact of the contaminated material surfaces and this equipment constitute an increasing source of infectious biological waste. Furthermore, infected people can transmit microbial infections easily because the protective equipment do not inactivate the microbial load generated while breathing, sneezing or coughing. In this regard, the goal of this work consisted of fabricating a transparent face shield with intrinsic antimicrobial activity that could provide extra-protection against infectious agents and reduce the generation of infectious waste. Thus, a single-use transparent antimicrobial face shield composed of polyethylene terephthalate and an antimicrobial coating of benzalkonium chloride has been developed for the next generation of facial protective equipment. The antimicrobial coating was analyzed by atomic force microscopy and field emission scanning electron microscopy with elemental analysis. This is the first facial transparent protective material capable of inactivating enveloped viruses such as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in less than one minute of contact, and the methicillin-resistant Staphylococcus aureus and Staphylococcus epidermidis. Bacterial infections contribute to severe pneumonia associated with the SARS-CoV-2 infection, and their resistance to antibiotics is increasing. Our extra protective broad-spectrum antimicrobial composite material could also be applied for the fabrication of other facial protective tools such as such as goggles, helmets, plastic masks and space separation screens used for counters or vehicles. This low-cost technology would be very useful to combat the current pandemic and protect health care workers from multidrug-resistant infections in developed and underdeveloped countries.

A novel aqueous extract from rice fermented with Aspergillus oryzae and Saccharomyces cerevisiae possesses an anti-influenza A virus activity.

Human influenza virus infections occur annually worldwide and are associated with high morbidity and mortality. Hence, development of novel anti-influenza drugs is urgently required. Rice Power® extract developed by the Yushin Brewer Co. Ltd. is a novel aqueous extract of rice obtained via saccharization and fermentation with various microorganisms, such as Aspergillus oryzae, yeast [such as Saccharomyces cerevisiae], and lactic acid bacteria, possessing various biological and pharmacological properties. In our previous experimental screening with thirty types of Rice Power® extracts, we observed that the 30th Rice Power® (Y30) extract promoted the survival of influenza A virus-infected Madin-Darby canine kidney (MDCK) cells. Therefore, to identify compounds for the development of novel anti-influenza drugs, we aimed to investigate whether the Y30 extract exhibits anti-influenza A virus activity. In the present study, we demonstrated that the Y30 extract strongly promoted the survival of influenza A H1N1 Puerto Rico 8/34 (A/PR/8/34), California 7/09, or H3N2 Aichi 2/68 (A/Aichi/2/68) viruses-infected MDCK cells and inhibited A/PR/8/34 or A/Aichi/2/68 viruses infection and growth in the co-treatment and pre-infection experiments. The pre-treatment of Y30 extract on MDCK cells did not induce anti-influenza activity in the cell. The Y30 extract did not significantly affect influenza A virus hemagglutination, and neuraminidase and RNA-dependent RNA polymerase activities. Interestingly, the electron microscopy experiment revealed that the Y30 extract disrupts the integrity of influenza A virus particles by permeabilizing the viral membrane envelope, suggesting that Y30 extract has a direct virucidal effect against influenza A virus. Furthermore, we observed that compared to the ethyl acetate (EtOAc) extract, the water extract of Y30 extract considerably promoted the survival of cells infected with A/PR/8/34 virus. These results indicated that more anti-influenza components were present in the water extract of Y30 extract than in the EtOAc extract. Our results highlight the potential of a rice extract fermented with A. oryzae and S. cerevisiae as an anti-influenza medicine and a drug source for the development of anti-influenza compounds.

Resistance of SARS-CoV-2 variants to neutralization by antibodies induced in convalescent patients with COVID-19.

Administration of convalescent plasma or neutralizing monoclonal antibodies (mAbs) is a potent therapeutic option for coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. However, SARS-CoV-2 variants with mutations in the spike protein have emerged in many countries. To evaluate the efficacy of neutralizing antibodies induced in convalescent patients against emerging variants, we isolate anti-spike mAbs from two convalescent COVID-19 patients infected with prototypic SARS-CoV-2 by single-cell sorting of immunoglobulin-G-positive (IgG+) memory B cells. Anti-spike antibody induction is robust in these patients, and five mAbs have potent neutralizing activities. The efficacy of most neutralizing mAbs and convalescent plasma samples is maintained against B.1.1.7 and mink cluster 5 variants but is significantly decreased against variants B.1.351 from South Africa and P.1 from Brazil. However, mAbs with a high affinity for the receptor-binding domain remain effective against these neutralization-resistant variants. Rapid spread of these variants significantly impacts antibody-based therapies and vaccine strategies against SARS-CoV-2.

Amplification-free RNA detection with CRISPR-Cas13.

CRISPR-based nucleic-acid detection is an emerging technology for molecular diagnostics. However, these methods generally require several hours and could cause amplification errors, due to the pre-amplification of target nucleic acids to enhance the detection sensitivity. Here, we developed a platform that allows "CRISPR-based amplification-free digital RNA detection (SATORI)", by combining CRISPR-Cas13-based RNA detection and microchamber-array technologies. SATORI detected single-stranded RNA targets with maximal sensitivity of ~10 fM in <5 min, with high specificity. Furthermore, the simultaneous use of multiple different guide RNAs enhanced the sensitivity, thereby enabling the detection of the SARS-CoV-2 N-gene RNA at ~5 fM levels. Therefore, we hope SATORI will serve as a powerful class of accurate and rapid diagnostics.

Modeling SARS-CoV-2 infection and its individual differences with ACE2-expressing human iPS cells.

Genetic differences are a primary reason for differences in the susceptibility and severity of COVID-19. As induced pluripotent stem (iPS) cells maintain the genetic information of the donor, they can be used to model individual differences in SARS-CoV-2 infection in vitro. We found that human iPS cells expressing the SARS-CoV-2 receptor angiotensin-converting enzyme 2 (ACE2) (ACE2-iPS cells) can be infected w SARS-CoV-2. In infected ACE2-iPS cells, the expression of SARS-CoV-2 nucleocapsid protein, budding of viral particles, and production of progeny virus, double membrane spherules, and double-membrane vesicles were confirmed. We performed SARS-CoV-2 infection experiments on ACE2-iPS/ embryonic stem (ES) cells from eight individuals. Male iPS/ES cells were more capable of producing the virus compared with female iPS/ES cells. These findings suggest that ACE2-iPS cells can not only reproduce individual differences in SARS-CoV-2 infection in vitro but also are a useful resource to clarify the causes of individual differences in COVID-19 due to genetic differences.

Protective Face Mask Filter Capable of Inactivating SARS-CoV-2, and Methicillin-Resistant Staphylococcus aureus and Staphylococcus epidermidis.

Face masks have globally been accepted to be an effective protective tool to prevent bacterial and viral transmission, especially against indoor aerosol transmission. However, commercial face masks contain filters that are made of materials that are not capable of inactivating either SARS-CoV-2 or multidrug-resistant bacteria. Therefore, symptomatic and asymptomatic individuals can infect other people even if they wear them because some viable viral or bacterial loads can escape from the masks. Furthermore, viral or bacterial contact transmission can occur after touching the mask, which constitutes an increasing source of contaminated biological waste. Additionally, bacterial pathogens contribute to the SARS-CoV-2-mediated pneumonia disease complex, and their resistance to antibiotics in pneumonia treatment is increasing at an alarming rate. In this regard, herein, we report the development of a non-woven face mask filter fabricated with a biofunctional coating of benzalkonium chloride that is capable of inactivating more than 99% of SARS-CoV-2 particles in one minute of contact, and the life-threatening methicillin-resistant Staphylococcus aureus and Staphylococcus epidermidis (normalized antibacterial halos of 0.52 ± 0.04 and 0.72 ± 0.04, respectively). Nonetheless, despite the results obtained, further studies are needed to ensure the safety and correct use of this technology for the mass production and commercialization of this broad-spectrum antimicrobial face mask filter. Our novel protective non-woven face mask filter would be useful for many healthcare workers and researchers working in this urgent and challenging field.