Publications by authors named "Akihide Ryo"

181 Publications

HBx-induced degradation of Smc5/6 complex impairs homologous recombination-mediated repair of damaged DNA.

J Hepatol 2021 Aug 25. Epub 2021 Aug 25.

Department of Gastroenterology, Graduate School of Medicine, The University of Tokyo, Tokyo 113-8655, Japan.

Background & Aims: Hepatitis B virus (HBV) causes hepatocellular carcinoma (HCC). While the need for HBV regulatory protein X (HBx) for viral transcription via impairment of the structural maintenance of chromosome 5/6 (Smc5/6) complex was recently demonstrated, HBx is also a potent driver of HCC. However, the mechanism by which HBx expression induces hepatocarcinogenesis is unclear.

Methods: Degradation of Smc5/6 complex and accumulation of DNA damage were observed in both in vivo and in vitro HBV infection models. Rescue experiments were performed using nitazoxanide (NTZ), which inhibits degradation of the Smc5/6 complex by HBx.

Results: Degradation of the Smc5/6 complex triggered by HBx impaired homologous recombination (HR) repair of DNA double-strand breaks (DSBs), leading to cellular transformation. We found that DNA damage accumulated in the liver tissue of HBV-infected humanized chimeric mice, HBx-transgenic mice, and human tissues. HBx suppressed the HR repair of DSBs, including that induced by the clustered regularly interspaced short palindromic repeats-Cas9 system, in an Smc5/6-dependent manner, which was rescued by restoring the Smc5/6 complex. NTZ restored HR repair in, and colony formation by, HBx-expressing cells.

Conclusions: Degradation of the Smc5/6 complex by HBx increases viral transcription and promotes cellular transformation by impairing HR repair of DSBs.

Lay Summary: HBV regulatory protein X (HBx) degrades the structural maintenance of chromosome 5/6 (Smc5/6) complex, which is required for viral replication. We found that degradation of the Smc5/6 complex also plays a key role in the accumulation of DNA damage, as well as in HBx-mediated tumorigenesis.
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http://dx.doi.org/10.1016/j.jhep.2021.08.010DOI Listing
August 2021

All-Trans Retinoic Acid Exhibits Antiviral Effect against SARS-CoV-2 by Inhibiting 3CLpro Activity.

Viruses 2021 08 23;13(8). Epub 2021 Aug 23.

Department of Microbiology, Yokohama City University Graduate School of Medicine, Yokohama 236-0004, Japan.

The pandemic of COVID-19 caused by SARS-CoV-2 continues to spread despite the global efforts taken to control it. The 3C-like protease (3CLpro), the major protease of SARS-CoV-2, is one of the most interesting targets for antiviral drug development because it is highly conserved among SARS-CoVs and plays an important role in viral replication. Herein, we developed high throughput screening for SARS-CoV-2 3CLpro inhibitor based on AlphaScreen. We screened 91 natural product compounds and found that all-trans retinoic acid (ATRA), an FDA-approved drug, inhibited 3CLpro activity. The 3CLpro inhibitory effect of ATRA was confirmed in vitro by both immunoblotting and AlphaScreen with a 50% inhibition concentration (IC) of 24.7 ± 1.65 µM. ATRA inhibited the replication of SARS-CoV-2 in VeroE6/TMPRSS2 and Calu-3 cells, with IC = 2.69 ± 0.09 µM in the former and 0.82 ± 0.01 µM in the latter. Further, we showed the anti-SARS-CoV-2 effect of ATRA on the currently circulating variants of concern (VOC); alpha, beta, gamma, and delta. These results suggest that ATRA may be considered as a potential therapeutic agent against SARS-CoV-2.
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http://dx.doi.org/10.3390/v13081669DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8402917PMC
August 2021

Rapid detection of neutralizing antibodies to SARS-CoV-2 variants in post-vaccination sera.

J Mol Cell Biol 2021 Aug 27. Epub 2021 Aug 27.

Department of Microbiology, Yokohama City University School of Medicine, Yokohama, Japan.

The uncontrolled spread of the COVID-19 pandemic has led to the emergence of different SARS-CoV-2 variants across the globe. The ongoing global vaccination strategy to curtail the COVID-19 juggernaut, is threatened by the rapidly spreading Variants of Concern (VOC) and other regional mutants, which are less responsive to neutralization by infection or vaccine derived antibodies. We have previously developed the hiVNT system which detects SARS-CoV-2 neutralizing antibodies in sera in less than three hours. In this study, we modify the hiVNT for rapid qualitative screening of neutralizing antibodies (nAb) to multiple VOC of SARS-CoV-2, and assess the neutralizing efficacy of the BNT162b2 mRNA vaccine on seven epidemiologically relevant SARS-CoV-2 variants. Here we show that the BNT162b2 mRNA vaccine can activate humoral immunity against the major SARS-CoV-2 mutants that are currently in circulation. Albeit a small sample size, we observed that one dose of vaccine was sufficient to elicit a protective humoral response in previously infected people. Using a panel of seven SARS-CoV-2 variants and a single prototype virus, our modified hiVNT would be useful for large-scale community wide testing to detect protective immunity that may confer vaccine/immune passport in the ongoing COVID-19 pandemic.
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http://dx.doi.org/10.1093/jmcb/mjab050DOI Listing
August 2021

Evaluation of a combination protocol of CT-first triage and active telemedicine methods by a selected team tackling COVID-19: An experimental research study.

J Infect Public Health 2021 Sep 18;14(9):1212-1217. Epub 2021 Aug 18.

Team COVID-19, Yokohama City University Hospital, 3-9 Fukuura, Kanazawa, Yokohama 2360004, Japan; Department of Gastroenterology, Yokohama City University Graduate School of Medicine, 3-9 Fukuura, Kanazawa, Yokohama 2360004, Japan.

Background: Many health care workers around the world tackled with COVID-19, however sadly, the infection of many medical care workers were reported. To reduce the risk of infection, we launched selected team (Team COVID) of non-specialists and brought in active telemedicine method and computed tomography (CT)-first protocol. We describe our actual practice and the health status of medical doctors dealing with COVID-19 patients.

Methods: Between April 17, 2020 and May 24, 2020, 10 doctors worked with COVID-19 patients as part of Team COVID. The Team COVID doctors used a CT-first triage protocol for outpatients and telemedicine for inpatients and outpatients. We evaluated paired serum-specific antibodies for SARS-CoV-2 at the initial and end of the study duration and PCR results for SARS-CoV-2 at the end of the study duration. Furthermore, 36-item short-form of the Medical Outcome Study Questionnaire (SF-36) at the beginning and end of the study period were evaluated.

Results: Ten doctors worked as Team COVID: seven internal medicine doctors and three surgeons. During the study period, Team COVID treated 165 individuals in the outpatient clinic and isolated hospitalized patients for 315 person-days. There were no positive results of serum-specific antibody testing and PCR testing for SARS-CoV-2 in Team COVID doctors. Furthermore, the SF-36 showed no deterioration in physical and mental QOL status. No in-hospital infection occurred during the study period.

Conclusions: The Team COVID fulfilled the treatment using the active telemedicine and CT-first triage protocol without in hospital infection and excess stress. The combination strategy seems acceptable for both the protection and stress relief among the medical staff.
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http://dx.doi.org/10.1016/j.jiph.2021.08.016DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8372458PMC
September 2021

Inhibitory effect of a novel thiazolidinedione derivative on hepatitis B virus entry.

Antiviral Res 2021 Oct 19;194:105165. Epub 2021 Aug 19.

Department of Microbiology, Faculty of Medicine, Graduate Faculty of Interdisciplinary Research, University of Yamanashi, Yamanashi, 409-3898, Japan. Electronic address:

The development of novel antivirals to treat hepatitis B virus (HBV) infection is still needed because currently available drugs do not completely eradicate chronic HBV in some patients. Recently, troglitazone and ciglitazone, classified among the compounds including the thiazolidinedione (TZD) moiety, were found to inhibit HBV infection, but these compounds are not clinically available. In this study, we synthesized 11 TZD derivatives, compounds 1-11, and examined the effect of each compound on HBV infection in HepG2 cells expressing NTCP (HepG2/NTCP cells). Among the derivatives, (Z)-5-((4'-(naphthalen-1-yl)-[1,1'-biphenyl]-4-yl)methylene)thiazolidine-2,4-dione (compound 6) showed the highest antiviral activity, with an IC value of 0.3 μM and a selectivity index (SI) of 85, but compound 6 did not affect HCV infection. Treatment with compound 6 inhibited HBV infection in primary human hepatocytes (PHHs) but did not inhibit viral replication in HepG2.2.15 cells or HBV DNA-transfected Huh7 cells. Moreover, treatment with compound 6 significantly impaired hepatitis delta virus (HDV) infection and inhibited a step in HBV particle internalization but did not inhibit attachment of the preS1 lipopeptide or viral particles to the cell surface. These findings suggest that compound 6 interferes with HBV infection via inhibition of the internalization process.
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http://dx.doi.org/10.1016/j.antiviral.2021.105165DOI Listing
October 2021

Combining IL-6 and SARS-CoV-2 RNAaemia-based risk stratification for fatal outcomes of COVID-19.

PLoS One 2021 11;16(8):e0256022. Epub 2021 Aug 11.

Department of Emergency Medicine, Yokohama City University Graduate School of Medicine, Yokohama, Kanagawa, Japan.

Background: The coronavirus disease 2019 (COVID-19) pandemic rapidly increases the use of mechanical ventilation (MV). Such cases further require extracorporeal membrane oxygenation (ECMO) and have a high mortality.

Objective: We aimed to identify prognostic biomarkers pathophysiologically reflecting future deterioration of COVID-19.

Methods: Clinical, laboratory, and outcome data were collected from 102 patients with moderate to severe COVID-19. Interleukin (IL)-6 level and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA copy number in plasma were assessed with ELISA kit and quantitative PCR.

Results: Twelve patients died or required ECMO owing to acute respiratory distress syndrome despite the use of MV. Among various variables, a ratio of oxygen saturation to fraction of inspired oxygen (SpO2/FiO2), IL-6, and SARS-CoV-2 RNA on admission before intubation were strongly predictive of fatal outcomes after the MV use. Moreover, among these variables, combining SpO2/FiO2, IL-6, and SARS-CoV-2 RNA showed the highest accuracy (area under the curve: 0.934). In patients with low SpO2/FiO2 (< 261), fatal event-rate after the MV use at the 30-day was significantly higher in patients with high IL-6 (> 49 pg/mL) and SARS-CoV-2 RNAaemia (> 1.5 copies/μL) compared to those with high IL-6 or RNAaemia or without high IL-6 and RNAaemia (88% vs. 22% or 8%, log-rank test P = 0.0097 or P < 0.0001, respectively).

Conclusions: Combining SpO2/FiO2 with high IL-6 and SARS-CoV-2 RNAaemia which reflect hyperinflammation and viral overload allows accurately and before intubation identifying COVID-19 patients at high risk for ECMO use or in-hospital death despite the use of MV.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0256022PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8357172PMC
August 2021

Highly specific monoclonal antibodies and epitope identification against SARS-CoV-2 nucleocapsid protein for antigen detection tests.

Cell Rep Med 2021 Jun 16;2(6):100311. Epub 2021 May 16.

Department of Microbiology, Yokohama City University School of Medicine, Yokohama, Kanagawa 236-0004, Japan.

The ongoing coronavirus disease 2019 (COVID-19) pandemic is a major global public health concern. Although rapid point-of-care testing for detecting viral antigen is important for management of the outbreak, the current antigen tests are less sensitive than nucleic acid testing. In our current study, we produce monoclonal antibodies (mAbs) that exclusively react with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and exhibit no cross-reactivity with other human coronaviruses, including SARS-CoV. Molecular modeling suggests that the mAbs bind to epitopes present on the exterior surface of the nucleocapsid, making them suitable for detecting SARS-CoV-2 in clinical samples. We further select the optimal pair of anti-SARS-CoV-2 nucleocapsid protein (NP) mAbs using ELISA and then use this mAb pair to develop immunochromatographic assay augmented with silver amplification technology. Our mAbs recognize the variants of concern (501Y.V1-V3) that are currently in circulation. Because of their high performance, the mAbs of this study can serve as good candidates for developing antigen detection kits for COVID-19.
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http://dx.doi.org/10.1016/j.xcrm.2021.100311DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8126173PMC
June 2021

Sustained Neutralizing Antibodies 6 Months Following Infection in 376 Japanese COVID-19 Survivors.

Front Microbiol 2021 7;12:661187. Epub 2021 May 7.

Department of Health Data Science, Yokohama City University Graduate School of Data Science, Yokohama, Japan.

There is scarce evidence regarding the long-term persistence of neutralizing antibodies among coronavirus disease 2019 (COVID-19) survivors. This study determined neutralizing antibody titers (NT) and antibodies against spike protein (SP) or nucleocapsid protein (NP) antigens approximately 6 months after the diagnosis of COVID-19. COVID-19 survivors in Japan were recruited. Serum samples and data related to patients' characteristics and COVID-19 history were collected. NT and titers of antibodies against NP and SP antigens were measured at 20-32 weeks after the first positive severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) test results. Factors associated with NT were identified using the multivariable linear regression and the correlations among NT and titers of immunoglobulin G (IgG) and total immunoglobulins (Igs) against NP and SP were assessed by Spearman's correlation. Among 376 participants (median [range] days after testing positive for SARS-CoV-2, 180 (147-224); median [range] years of age, 50 (20-78); 188 [50%] male), most tested positive for NT ( = 367, 98%), SP-IgG ( = 344, 91%), SP-total Ig ( = 369, 98%), NP-IgG ( = 314, 84%), and NP-total Ig ( = 365, 97%). Regression analysis indicated that higher BMI, fever, and the requirement of mechanical ventilation or extracorporeal membrane oxygenation were significantly associated with higher NT. Anti-SP antibodies correlated moderately with NT (Spearman's correlation: 0.63 for SP IgG; 0.57 for SP-total Ig), while the correlation was weak for anti-NP antibodies (0.37 for NP IgG; 0.32 for NP-total Ig). Most COVID-19 survivors had sustained neutralizing antibodies and tested positive for SP-total Ig and NP-total Ig approximately 6 months after infection.
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http://dx.doi.org/10.3389/fmicb.2021.661187DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8137897PMC
May 2021

Cleavage of TANK-Binding Kinase 1 by HIV-1 Protease Triggers Viral Innate Immune Evasion.

Front Microbiol 2021 27;12:643407. Epub 2021 Apr 27.

Department of Microbiology, Yokohama City University School of Medicine, Yokohama, Japan.

Type-I interferons (IFN-I) are the innate immune system's principal defense against viral infections. Human immunodeficiency virus-1 (HIV-1) has evolved several ways to suppress or evade the host's innate immunity in order to survive and replicate to sustain infection. Suppression of IFN-I is one among the multiple escape strategies used by HIV-1 to prevent its clearance. HIV-1 protease which helps in viral maturation has also been observed to cleave host cellular protein kinases. In this study we performed a comprehensive screening of a human kinase library using AlphaScreen assay and identified that TANK binding kinase-1 (TBK1) was cleaved by HIV-1 protease (PR). We demonstrate that PR cleaved TBK1 fails to phosphorylate IFN regulatory factor 3 (IRF3), thereby reducing the IFN-I promoter activity and further reveal that the PR mediated suppression of IFN-I could be counteracted by protease inhibitors (PI) . We have also revealed that mutations of HIV-1 PR that confer drug resistance to PIs reduce the enzyme's ability to cleave TBK1. The findings of this study unearth a direct link between HIV-1 PR activity and evasion of innate immunity by the virus, the possible physiological relevance of which warrants to be determined.
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http://dx.doi.org/10.3389/fmicb.2021.643407DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8110901PMC
April 2021

Establishment of a Cell Culture Model Permissive for Infection by Hepatitis B and C Viruses.

Hepatol Commun 2021 04 19;5(4):634-649. Epub 2020 Dec 19.

Department of Microbiology Graduate School of Medical Science University of Yamanashi Yamanashi Japan.

Compared with each monoinfection, coinfection with hepatitis B virus (HBV) and hepatitis C virus (HCV) is well known to increase the risks of developing liver cirrhosis and hepatocellular carcinoma. However, the mechanism by which HBV/HCV coinfection is established in hepatocytes is not well understood. Common cell culture models for coinfection are required to examine viral propagation. In this study, we aimed to establish a cell line permissive for both HBV and HCV infection. We first prepared a HepG2 cell line expressing sodium taurocholate cotransporting polypeptide, an HBV receptor, and then selected a cell line highly permissive for HBV infection, G2/NT18-B. After transduction with a lentivirus-encoding microRNA-122, the cell line harboring the highest level of replicon RNA was selected and then treated with anti-HCV compounds to eliminate the replicon RNA. The resulting cured cell line was transduced with a plasmid-encoding CD81. The cell line permissive for HCV infection was cloned and then designated the G2BC-C2 cell line, which exhibited permissiveness for HBV and HCV propagation. JAK inhibitor I potentiated the HCV superinfection of HBV-infected cells, and fluorescence-activated cell-sorting analysis indicated that HBV/HCV double-positive cells accounted for approximately 30% of the coinfected cells. Among several host genes tested, cyclooxygenase-2 showed synergistic induction by coinfection compared with each monoinfection. These data indicate that our HBV/HCV coinfection system provides an easy-to-use platform for the study of host and viral responses against coinfection and the development of antiviral agents targeting HBV and HCV.
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http://dx.doi.org/10.1002/hep4.1653DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8034569PMC
April 2021

Carbonic anhydrase 13 suppresses bone metastasis in breast cancer.

Cancer Treat Res Commun 2021 9;27:100332. Epub 2021 Feb 9.

Department of Biochemistry, The Jikei University School of Medicine, 3-25-8 Nishi-shinbashi, Minato-ku, Tokyo 105-8461, Japan. Electronic address:

Metastatic progression is the leading cause of mortality in breast cancer. However, molecular mechanisms that govern this process remain unclear. In this study, we found that carbonic anhydrase 13 (CA13) plays a potential role in suppressing bone metastasis. iRFP713-labeled iCSCL-10A (iRFP-iCSCL-10A) breast cancer cells, which exhibit the hallmarks of cancer stem cells, exerted the ability of bone metastasis in hind legs after 5-week injections, whereas no metastasis was observed in control iRFP713-labeled MCF-10A (iRFP-MCF10A) cells. Transcriptome analysis indicated that the expression of several genes, including metabolism-related CA13, was reduced in bone metastatic iRFP-iCSCL-10A cells. In vitro and in vivo analyses demonstrated that overexpression of CA13 in iRFP-iCSCL-10A cells suppressed migration, invasion, and bone metastasis, together with the reduction of VEGF-A and M-CSF expression. Furthermore, we found that breast cancer patients with a low CA13 expression had significantly shorter overall survival and disease-free survival rates compared to those with higher CA13 expression. These findings suggest that CA13 may act as a novel prognostic biomarker and would be a therapeutic candidate for the prevention of bone metastasis in breast cancer.
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http://dx.doi.org/10.1016/j.ctarc.2021.100332DOI Listing
February 2021

Severe acute respiratory syndrome coronavirus 2 prevalence in saliva and gastric and intestinal fluid in patients undergoing gastrointestinal endoscopy in coronavirus disease 2019 endemic areas: Prospective cross-sectional study in Japan.

Dig Endosc 2021 Feb 6. Epub 2021 Feb 6.

Department of, Gastroenterology and Hepatology, Graduate School of Medicine, Yokohama City University, Kanagawa, Japan.

Objectives: Gastrointestinal endoscopy (GIE) is useful for the early detection and treatment of many diseases; however, GIE is considered a high-risk procedure in the coronavirus disease 2019 (COVID-19) pandemic era. This study aimed to explore the rate of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) positivity in saliva and gastrointestinal fluids to which endoscopy medical staff are exposed.

Methods: The study was a single-center cross-sectional study. From June 1 to July 31, 2020, all patients who underwent GIE at Yokohama City University Hospital were registered. All patients provided 3 mL of saliva. For upper GIE, 10 mL of gastric fluid was collected through the endoscope. For lower GIE, 10 mL of intestinal fluid was collected through the endoscope. The primary outcome was the positive rate of SARS-CoV-2 in saliva and gastrointestinal fluids. We also analyzed serum-specific antibodies for SARS-CoV-2 and patients' background information.

Results: A total of 783 samples (560 upper GIE and 223 lower GIE samples) were analyzed. Polymerase chain reaction (PCR) on saliva samples did not show any positive results in either upper or lower GIE samples. However, 2.0% (16/783) of gastrointestinal fluid samples tested positive for SARS-CoV-2. No significant differences in age, sex, purpose of endoscopy, medication, or rate of antibody test positivity were found between PCR positive and PCR negative cases.

Conclusions: Asymptomatic patients, even those with no detectable virus in their saliva, had SARS-CoV-2 in their gastrointestinal tract. Endoscopy medical staff should be aware of infection when performing procedures. The study was registered as UMIN000040587.
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http://dx.doi.org/10.1111/den.13945DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8014498PMC
February 2021

Development of an Automated Chemiluminescence Assay System for Quantitative Measurement of Multiple Anti-SARS-CoV-2 Antibodies.

Front Microbiol 2020 15;11:628281. Epub 2021 Jan 15.

Department of Microbiology, Yokohama City University Graduate School of Medicine, Yokohama, Japan.

Objectives: Serological tests for COVID-19 have been instrumental in studying the epidemiology of the disease. However, the performance of the currently available tests is plagued by the problem of variability. We have developed a high-throughput serological test capable of simultaneously detecting total immunoglobulins (Ig) and immunoglobulin G (IgG) against nucleocapsid protein (NP) and spike protein (SP) and report its performance in detecting COVID-19 in clinical samples.

Methods: We designed and prepared reagents for measuring NP-IgG, NP-Total Ig, SP-IgG, and SP-Total Ig (using N-terminally truncated NP (ΔN-NP) or receptor-binding domain (RBD) antigen) dedicated automated chemiluminescent enzyme immunoassay analyzer AIA-CL1200. After determining the basal thresholds based on 17 sera obtained from confirmed COVID-19 patients and 600 negative sera, the clinical validity of the assay was evaluated using independent 202 positive samples and 1,000 negative samples from healthy donors.

Results: All of the four test parameters showed 100% specificity individually (1,000/1,000; 95%CI, 99.63-100). The sensitivity of the assay increased proportionally to the elapsed time from symptoms onset, and all the tests achieved 100% sensitivity (153/153; 95%CI, 97.63-100) after 13 days from symptoms onset. NP-Total Ig was the earliest to attain maximal sensitivity among the other antibodies tested.

Conclusion: Our newly developed serological testing exhibited 100% sensitivity and specificity after 13 days from symptoms onset. Hence, it could be used as a reliable method for accurate detection of COVID-19 patients and to evaluate seroprevalence and possibly for surrogate assessment of herd immunity.
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http://dx.doi.org/10.3389/fmicb.2020.628281DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7843449PMC
January 2021

Editorial for the Special Issue: Molecular Epidemiology, Diagnostics and Management of Respiratory Virus Infections.

Microorganisms 2020 Dec 20;8(12). Epub 2020 Dec 20.

Department of Microbiology, School of Medicine, Yokohama City University, 3-9 Fukuura, Kanazawa-ku, Yokohama-shi, Kanagawa 236-0004, Japan.

In Japan, there is a proverb that the common cold is associated with all diseases [...].
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http://dx.doi.org/10.3390/microorganisms8122041DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7766214PMC
December 2020

A Longitudinal Study on Enteric Virus Contamination in Bivalves along the Coast of Ibaraki Prefecture, Japan.

J Food Prot 2021 Jun;84(6):946-952

Gunma Paz University Graduate School of Health Science, Gunma, Japan.

Abstract: During the 2014 to 2018 seasons, we conducted a longitudinal study involving enteric virus surveillance in bivalves, including natural oysters and clams harvested in Ibaraki Prefecture, Japan. Some norovirus (NoV) contaminations were detected in natural oysters, whereas no enteric virus was found in clams. NoVs detected in oysters were of the genotypes GII.4 and GII.6, both of which are closely related genetically to the NoV strains prevalent in humans. We found low level of enteric virus contamination in bivalves collected along the coast of Ibaraki Prefecture. The possibility of food poisoning caused by these viruses appears low, and few cases of infectious disease have been observed in the surrounding area. The harvest timing was more related to contamination quantity than the harvest area in many enteric viruses. Our results highlight that contamination of bivalves by enteric viruses may depend upon the prevalence of human diarrhea and illness.

Highlights:
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http://dx.doi.org/10.4315/JFP-20-353DOI Listing
June 2021

Point mutation in the stop codon of increases the growth rate of subspecies .

Microbiology (Reading) 2021 02;167(2)

Department of Mycobacteriology, Leprosy Research Center, National Institute of Infectious Diseases, Tokyo, Japan.

subspecies (MAH) is a pathogen that causes various non-tuberculous mycobacterial diseases in humans and animals worldwide. Among the genus, MAH is characterized by relatively slow growth. Here, we isolated a rapidly growing variant of the MAH 104 strain. The variant strain (named N104) exhibited an enhanced growth rate and higher motility compared to the parent MAH 104 strain (P104). Whole-genome sequencing analysis of N104 revealed the loss of the stop codon of due to a single nucleotide replacement, resulting in the substitution of the codon for tryptophan. Notably, exclusion of the stop codon ligated the open reading frames and caused the fusion of two adjacent proteins. A revertant parent strain, in which a mutation was introduced to restore the stop codon, revealed that elimination of the stop codon in was responsible for the N104 phenotype. Furthermore, we analysed the phenotypes of the parent and mutated strains by determining the functions of the and coding regions flanking the stop codon. The mutant strains, expected to express a fusion protein, exhibited increased resistance to antimicrobial drugs and exogenous copper toxicity compared to that of the parent strains. These findings suggest that the fusion of the - and -encoding regions in the mutant N104 strain could be related to the modified functions of these intrinsic proteins.
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http://dx.doi.org/10.1099/mic.0.001007DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8131024PMC
February 2021

Zika virus protease induces caspase-independent pyroptotic cell death by directly cleaving gasdermin D.

Biochem Biophys Res Commun 2021 01 15;534:666-671. Epub 2020 Nov 15.

Department of Microbiology, Yokohama City University School of Medicine, Kanagawa, 236-0004, Japan. Electronic address:

The association of Zika virus (ZIKV) infection with a congenital malformation in fetuses, neurological, and other systemic complications in adults have brought significant global health emergency. ZIKV targets nerve cells in the brain and causes cell death, such as pyroptosis, leading to neuroinflammation. Here we described a novel mechanism of pyroptosis caused by ZIKV protease. We found that ZIKV protease directly cleaved the GSDMD into N-terminal fragment (1-249) leading to pyroptosis in a caspase-independent manner, suggesting a direct mechanism of ZIKV-induced cell death and subsequent inflammation. Our findings might shed new light to explore the pathogenesis of ZIKV infections where ZIKV protease might be a suitable target for the development of antiviral agents.
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http://dx.doi.org/10.1016/j.bbrc.2020.11.023DOI Listing
January 2021

Treating COVID-19: are we missing out the window of opportunity?

J Antimicrob Chemother 2021 01;76(2):283-285

Department of Microbiology, Yokohama City University School of Medicine, Kanagawa, Japan.

Severe COVID-19 is a biphasic illness, with an initial viral replication phase, followed by a cascade of inflammatory events. Progression to severe disease is predominantly a function of the inflammatory cascade, rather than viral replication per se. This understanding can be effectively translated to changing our approach in managing the disease. The natural course of disease offers us separate windows of specific time intervals to administer either antiviral or immunomodulatory therapy. Instituting the right attack at the right time would maximize the benefit of treatment. This concept must also be factored into studies that assess the efficacy of antivirals and immunomodulatory agents against COVID-19.
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http://dx.doi.org/10.1093/jac/dkaa442DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7665528PMC
January 2021

Detailed Molecular Interactions of Favipiravir with SARS-CoV-2, SARS-CoV, MERS-CoV, and Influenza Virus Polymerases In Silico.

Microorganisms 2020 Oct 20;8(10). Epub 2020 Oct 20.

Department of Microbiology, Yokohama City University School of Medicine, Yokohama, Kanagawa 236-0004, Japan.

Favipiravir was initially developed as an antiviral drug against influenza and is currently used in clinical trials against severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infection (COVID-19). This agent is presumably involved in RNA chain termination during influenza virus replication, although the molecular interactions underlying its potential impact on the coronaviruses including SARS-CoV-2, SARS-CoV, and Middle East respiratory syndrome coronavirus (MERS-CoV) remain unclear. We performed in silico studies to elucidate detailed molecular interactions between favipiravir and the SARS-CoV-2, SARS-CoV, MERS-CoV, and influenza virus RNA-dependent RNA polymerases (RdRp). As a result, no interactions between favipiravir ribofuranosyl-5'-triphosphate (F-RTP), the active form of favipiravir, and the active sites of RdRps (PB1 proteins) from influenza A (H1N1)pdm09 virus were found, yet the agent bound to the tunnel of the replication genome of PB1 protein leading to the inhibition of replicated RNA passage. In contrast, F-RTP bound to the active sites of coronavirus RdRp in the presence of the agent and RdRp. Further, the agent bound to the replicated RNA terminus in the presence of agent, magnesium ions, nucleotide triphosphate, and RdRp proteins. These results suggest that favipiravir exhibits distinct mechanisms of action against influenza virus and various coronaviruses.
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http://dx.doi.org/10.3390/microorganisms8101610DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7589801PMC
October 2020

A Hyperactive RelA/p65-Hexokinase 2 Signaling Axis Drives Primary Central Nervous System Lymphoma.

Cancer Res 2020 12 16;80(23):5330-5343. Epub 2020 Oct 16.

Department of Neurosurgery, Massachusetts General Hospital, Boston, Massachusetts.

Primary central nervous system lymphoma (PCNSL) is an isolated type of lymphoma of the central nervous system and has a dismal prognosis despite intensive chemotherapy. Recent genomic analyses have identified highly recurrent mutations of MYD88 and CD79B in immunocompetent PCNSL, whereas LMP1 activation is commonly observed in Epstein-Barr virus (EBV)-positive PCNSL. However, a lack of clinically representative preclinical models has hampered our understanding of the pathogenic mechanisms by which genetic aberrations drive PCNSL disease phenotypes. Here, we establish a panel of 12 orthotopic, patient-derived xenograft (PDX) models from both immunocompetent and EBV-positive PCNSL and secondary CNSL biopsy specimens. PDXs faithfully retained their phenotypic, metabolic, and genetic features, with 100% concordance of MYD88 and CD79B mutations present in PCNSL in immunocompetent patients. These models revealed a convergent functional dependency upon a deregulated RelA/p65-hexokinase 2 signaling axis, codriven by either mutated MYD88/CD79B or LMP1 with Pin1 overactivation in immunocompetent PCNSL and EBV-positive PCNSL, respectively. Notably, distinct molecular alterations used by immunocompetent and EBV-positive PCNSL converged to deregulate RelA/p65 expression and to drive glycolysis, which is critical for intracerebral tumor progression and FDG-PET imaging characteristics. Genetic and pharmacologic inhibition of this key signaling axis potently suppressed PCNSL growth and . These patient-derived models offer a platform for predicting clinical chemotherapeutics efficacy and provide critical insights into PCNSL pathogenic mechanisms, accelerating therapeutic discovery for this aggressive disease. SIGNIFICANCE: A set of clinically relevant CNSL xenografts identifies a hyperactive RelA/p65-hexokinase 2 signaling axis as a driver of progression and potential therapeutic target for treatment and provides a foundational preclinical platform. GRAPHICAL ABSTRACT: http://cancerres.aacrjournals.org/content/canres/80/23/5330/F1.large.jpg.
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http://dx.doi.org/10.1158/0008-5472.CAN-20-2425DOI Listing
December 2020

Development of Monoclonal Antibodies and Antigen-Capture ELISA for Human Parechovirus Type 3.

Microorganisms 2020 Sep 19;8(9). Epub 2020 Sep 19.

Department of Microbiology, Yokohama City University School of Medicine, Kanagawa 236-0004, Japan.

Human parechovirus type 3 (HPeV3) is an etiologic agent of respiratory diseases, meningitis, and sepsis-like illness in both infants and adults. Monoclonal antibodies (mAbs) can be a promising diagnostic tool for antigenic diseases such as virus infection, as they offer a high specificity toward a specific viral antigen. However, to date, there is no specific mAb available for the diagnosis of HPeV3 infection. In this study, we developed and characterized mAbs specific for HPeV3 capsid protein VP0. We used cell-free, wheat germ-synthesized viral VP0 protein for immunizing BALB/c mice to generate hybridomas. From the resultant hybridoma clones, we selected nine clones producing mAbs reactive to the HPeV3-VP0 antigen, based on enzyme-linked immunosorbent assay (ELISA). Epitope mapping showed that these mAbs recognized three distinct domains in HPeV3 VP0. Six mAbs recognized HPeV3 specifically and the other three mAbs showed cross-reactivity with other HPeVs. Using the HPeV3-specific mAbs, we then developed an ELISA for viral antigen detection that could be reliably used for laboratory diagnosis of HPeV3. This ELISA system exhibited no cross-reactivity with other related viruses. Our newly developed mAbs would, thus, provide a useful set of tools for future research and ensure HPeV3-specific diagnosis.
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http://dx.doi.org/10.3390/microorganisms8091437DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7563955PMC
September 2020

Potent antiviral effect of silver nanoparticles on SARS-CoV-2.

Biochem Biophys Res Commun 2020 11 11;533(1):195-200. Epub 2020 Sep 11.

Department of Microbiology, Yokohama City University School of Medicine, Kanagawa, 236-0004, Japan. Electronic address:

The pandemic of COVID-19 is spreading unchecked due to the lack of effective antiviral measures. Silver nanoparticles (AgNP) have been studied to possess antiviral properties and are presumed to inhibit SARS-CoV-2. Due to the need for an effective agent against SARS-CoV-2, we evaluated the antiviral effect of AgNPs. We evaluated a plethora of AgNPs of different sizes and concentration and observed that particles of diameter around 10 nm were effective in inhibiting extracellular SARS-CoV-2 at concentrations ranging between 1 and 10 ppm while cytotoxic effect was observed at concentrations of 20 ppm and above. Luciferase-based pseudovirus entry assay revealed that AgNPs potently inhibited viral entry step via disrupting viral integrity. These results indicate that AgNPs are highly potent microbicides against SARS-CoV-2 but should be used with caution due to their cytotoxic effects and their potential to derange environmental ecosystems when improperly disposed.
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http://dx.doi.org/10.1016/j.bbrc.2020.09.018DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7486059PMC
November 2020

Molecular pharmacology of ciclesonide against SARS-CoV-2.

J Allergy Clin Immunol 2020 08 13;146(2):330-331. Epub 2020 Jun 13.

Department of Microbiology, Yokohama City University School of Medicine, Kanagawa, Japan.

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http://dx.doi.org/10.1016/j.jaci.2020.05.029DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7293530PMC
August 2020

promotes its own survival via choline-binding protein CbpC-mediated degradation of ATG14.

Autophagy 2020 08 17;16(8):1529-1531. Epub 2020 Jun 17.

Department of Bacteriology I, National Institute of Infectious Diseases , Tokyo, Japan.

Streptococcus Pneumoniae: is an opportunistic bacterial pathogen that can promote severe infection by overcoming the epithelial and blood-brain barrier. Pneumococcal cell-surface virulence factors, including cell wall-anchored choline-binding proteins (Cbps) play pivotal roles in promoting invasive disease. We reported previously that intracellular pneumococci were detected by hierarchical macroautophagic/autophagic processes that ultimately lead to bacterial elimination. However, whether intracellular pneumococci can evade autophagy by deploying Cbps remains unclear. In this study, we explore the biological functions of Cbps and reveal their roles in manipulating the autophagic process. Specifically, we found that CbpC-activated autophagy takes place via its interactions with ATG14 (autophagy related 14) and SQSTM1/p62 (sequestosome1). Importantly, CbpC dampens host autophagy by promoting ATG14 degradation via the ATG14-CbpC-SQSTM1/p62 axis. CbpC-induced reductions in ATG14 levels result in impaired ATG14-STX17 complex formation. In pneumococcal-infected cells, ATG14 levels are dramatically reduced in a CbpC-dependent manner that results in suppression of autophagy-mediated degradation and enhanced bacterial survival. Taken together, our results reveal a novel mechanism via which pneumococci can manipulate host autophagy responses, in this case, by employing CbpC as a trap to promote ATG14 depletion. Our findings highlight a novel and sophisticated tactic used by that serves to promote intracellular survival.
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http://dx.doi.org/10.1080/15548627.2020.1776475DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7480810PMC
August 2020

SIRT1 enhances hepatitis virus B transcription independent of hepatic autophagy.

Biochem Biophys Res Commun 2020 06 25;527(1):64-70. Epub 2020 Apr 25.

Department of Gastroenterology and Hepatology, Osaka University, Graduate School of Medicine, Suita, Japan. Electronic address:

Autophagy is an intracellular process that can lead to the degradation of malfunctioned proteins and damaged organelles to maintain homeostasis during cellular stress. Here, we evaluated the change in hepatitis B virus (HBV) production by regulating hepatic autophagy in HBV-producing cells. We examined focusing on a relation with a positive autophagy regulator, sirtuin1 (SIRT1). Starvation and rapamycin treatment induced autophagy with increasing SIRT1 protein, HBc protein and pregenomic RNA (pgRNA) levels in HBV- producing cells. Knockdown of Atg7 or Atg13 suppressed hepatic autophagy, and it did not change SIRT1 protein, HBc protein or pgRNA levels in HBV- producing cells. Resveratrol, which increases SIRT1 expression and activity, promoted autophagy and increased HBc protein and pgRNA levels. siRNA-mediated knockdown of SIRT1 inhibited autophagy and decreased HBc protein and pgRNA levels. In SIRT1-knockdown cells, starvation promoted autophagy but did not increase HBc protein and pgRNA levels. In conclusion, HBc protein and pgRNA levels are upregulated not by the autophagic process itself but by the SIRT1 expression level.
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http://dx.doi.org/10.1016/j.bbrc.2020.04.031DOI Listing
June 2020

Molecular evolution of the capsid () region in human norovirus genogroup II genotype 3.

Heliyon 2020 May 3;6(5):e03835. Epub 2020 May 3.

Department of Health Science, Gunma Paz University Graduate School of Health Sciences, 1-7-1 Tonyamachi, Takasaki-shi, Gunma 370-0006, Japan.

Norovirus GII.3 has been suggested to be a prevalent genotype in patients with acute gastroenteritis. However, the genetic properties of the region encoding the major GII.3 antigen remain unclear. Here, we performed molecular evolutionary analyses of the GII.3 region detected in various countries. We performed time-scaled phylogenetic analyses, selective pressure analyses, phylogenetic distance analyses, and conformational epitope analyses. The time-scaled phylogenetic tree showed that an ancestor of the GII.3 region diverged from the common ancestors of the GII.6, GII.11, GII.18, and GII.19 approximately 70 years ago with relatively low divergence. The evolutionary rate of the GII.3 region was rapid (4.82 × 10 substitutions/site/year). Furthermore, one positive site and many negative selection sites were observed in the capsid protein. These results suggest that the GII.3 region rapidly evolved with antigenic variations.
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http://dx.doi.org/10.1016/j.heliyon.2020.e03835DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7205756PMC
May 2020

Interpreting Diagnostic Tests for SARS-CoV-2.

JAMA 2020 Jun;323(22):2249-2251

Department of Microbiology, Yokohama City University, Yokohama, Japan.

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http://dx.doi.org/10.1001/jama.2020.8259DOI Listing
June 2020
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