Shuichi Mori

Analysis of anti-rotavirus activity of extract from Stevia rebaudiana

Anti-human rotavirus (HRV) activity of hot water extracts from Stevia rebaudiana (SE) was examined. SE inhibited the replication of all four serotypes of HRV in vitro. This inhibitory effect of SE was not reduced on the prior exposure of SE to HCl for 30 min at pH 2. Binding assay with radiolabeled purified viruses indicated that the inhibitory mechanism of SE is the blockade of virus binding. The SE inhibited the binding of anti-VP7 monoclonal antibody to HRV-infected MA104 cells. The inhibitory components of SE were found to be heterogeneous anionic polysaccharides with different ion charges. The component analyses suggested that the purified fraction named as Stevian with the highest inhibitory activity consists of the anionic polysaccharide with molecular weight of 9800, and contains Ser and Ala as amino acids. Analyses of sugar residues suggest uronic acid(s) as sugar components. It did not contain amino and neutral sugars and sulfate residues. These findings suggest that SE may bind to 37 kD VP7 and interfere with the binding of VP7 to the cellular receptors by steric hindrance, which results in the blockade of the virus attachment to cells.

Broad spectrum anti-RNA virus activities of titanium and vanadium substituted polyoxotungstates

Seven polyoxotungstates substituted with vanadium or titanium atoms were examined for their activity against Flaviviridae (Dengue fever virus, DFV), Orthomyxoviridae (influenza virus type A, fluV-A), Paramyxoviridae (respiratory syncytial virus, RSV, parainfluenza virus type 2, PfluV-2 and canine distemper virus, CDV) and Lentiviridae (human immunodeficiency virus type 1, HIV-1) families. Among the seven polyoxotungstates examined, PM-43 [K(5)[SiVW(11)O(40)]], PM-47 [K(7)[BVW(11)O(40)]], and PM-1001 [K(10)Na(VO)(3)(SbW(9)O(33))(2)]26H(2)O contained vanadium. PM-1002 had the same core structure of (VO)(3)(SbW(9)O(33))(2) as PM-1001; however, three V atoms of PM-1001 consisted of two V(IV) and one V(V) and those of PM-1002 consisted of three V(IV). On the other hand, PM-518 [[Et(2)NH(2)](7)[PTi(2)W(10)O(40)]], PM-520 [Pri(2)NH(2)](5)[PTiW(11)O(40)] and PM-523 [PriNH(3)](6)H[PTi(2)W(10)O(38)(O(2))(2)]H(2)O all contained titanium. All compounds showed broad spectrum antiviral activity against all viruses examined except for PMs-43, -518 and -523 which did not exhibit inhibitory activity at >/=50 microM against PfluV-2, CDV and DFV, respectively. All compounds were inhibitory against HIV replication at an EC(50) of less than 2.0 microM. Among them, PMs-1001 and -1002 showed the most potent inhibition. The compounds were not toxic for MDCK, HEp-2 and Vero cells at a concentration of 200 microM. For the exponentially growing MT-4 cells, the vanadium containing polyoxometalates (PMs-43, 47, 1001, 1002) showed toxicity at concentrations between 41 and 47 microM. On the other hand, titanium containing polyoxometalates (PMs-518, -520, -523) were not toxic at 100 microM. The mechanism of anti-HIV action of PM-1001 was analyzed: it affected the binding of HIV to the cell membrane and syncytium formation between HIV-infected and uninfected cells.

Antiviral activity of NMSO3 against respiratory syncytial virus infection in vitro and in vivo.

NMSO3, a sulfated sialyl lipid was evaluated for its efficacy against respiratory syncytial virus (RSV) and other myxovirus infections in cell culture. The median effective concentration (50% effective concentration, EC(50)) of NMSO3 against replication of the Long strain of RSV in HEp-2 cells was 0.2 and 0.32 microM by optical ELISA and the plaque reduction method, respectively. On the other hand, the corresponding values for ribavirin were 10.5 and 11.2 microM, respectively. NMSO3 showed potent activity against other laboratory strains as well as fresh clinical isolates of RSV, and the average EC(50) was similar to that for Long strain. NMSO3 exhibited minimal cytotoxicity against HEp-2, MDCK, HMV-2 and Vero cells for which the median cytotoxic concentration (CC(50)) was more than 685 microM. The selectivity index [SI=(CC(50) for HEp-2/EC(50))] of NMSO3 for RSV exceeded 2978 and that of ribavirin was 6. The EC(50) of NMSO3 against influenza virus (FluV) A (H3N2) was 23.8 by the MTT method using HMV-2 cells, and 17.8 microM by the TCID(50) method using MDCK cells. NMSO3 did not inhibit replication of influenza B virus, parainfluenza virus type 2 and canine distemper virus at 103 microM. NMSO3 inhibited RSV infection of HEp-2 cells when it was added between 0 and 1.5 h after virus infection. By a temperature shift experiment during the period of contact between the virus and cells, NMSO3 inhibited both the binding of RSV to the cells and its penetration into the cells. Prophylactic and therapeutic efficacy of NMSO3 against RSV infection in cotton rats was examined. Intraperitoneal administration of 100 mg/kg per day of NMSO3 to cotton rats from 1 day before or 1 h after to 3 days after the RSV infection, once a day every day, decreased the RSV titer in lungs to 10(-1.26) to 10(-1.63) compared to the control rats which were infected with RSV and left untreated.

Anti-RNA virus activity of polyoxometalates.

Abstract The anti-RNA virus activity of polyoxometalates (POM) is reviewed, with a special emphasis on the anti-respiratory virus activities. There are many causative agents of acute viral respiratory infections; and it is rather difficult to identify the relevant agent in a given case by rapid clinical means. During acute progress of infection before the definitive diagnosis is obtained physicians need to prescribe certain broad spectrum anti-viral drugs. A titanium containing polyoxotungstate, PM-523 exhibited potent anti-influenza virus (FluV) A and anti-respiratory syncytial virus (RSV) activities in vitro. Therapeutic effect of FluV A infected mice with aerosol inhalation of PM-523 was proven. A vanadium substituted polyoxotungstate, PM-1001 has antiviral activity against FluV A, RSV, parainfluenza virus (PfluV) type 2, Dengue fiver virus, HIV-1 and SARS coronavirus in vitro. Thus, POMs have been proven to be broad spectrum and non-toxic anti-RNA virus agents in both in vitro and in vivo experiments and are promising candidates for first-line therapeutics in acute respiratory diseases.

Role of MBL-associated Serine Protease (MASP) On Activation of the Lectin Complement Pathway

Mannose-binding lectin (MBL) and ficolin are pattern recognition molecules in the complex with the MBL-associated serine proteases (MASPs). Three kinds of MASPs, termed as MASP-1, MASP-2 and MASP-3 have been identified. When MBL or ficolins binds to carbohydrates on the surface of microbes, conformational modifications of these molecules trigger to activate zymogens of MASPs, followed by consequential complement activation. MASP-2 cleaves C4 and C2 to make a C3 convertase, C4b2a. MASP-1 has an ability to cleave C3 directly, although this activity has not been detected in physiological conditions. Natural target molecules for MASP-3 are still discussible. To elucidate the physiological meanings of MASPs, we generated MASPs-deficient mice. Not only MASP-2-deficient mouse but also MASP-1-/MASP-3-deficient mouse reduced activities for C3 deposition on the surface of mannan and zymosan, suggesting MASP-1/3 also contribute the activation of complement by the lectin pathway. Also, MASP-1/3-deficient mice showed the susceptible to an influenza virus.

In vitro Antimyxovirus and Anti-Human Immunodeficiency Virus Activities of Polyoxometalates

Polyoxometalates have been shown to inhibit the replication of retro-, toga-, paramyxo- and herpesviruses. The primary mechanism of the anti-human immunodeficiency virus type 1 (HIV-1) action of polyoxometalates seems to be inhibition of binding of virus to cells and inhibition of syncytium formation. Since myxoviruses and HIV-1 are known to interact with the cytoplasmic membrane by adsorption and penetration of virus and by fusion of infected and uninfected cells, 25 polyoxometalates were examined for anti-ortho-, anti-paramyxovirus and anti-HIV-1 activity in vitro. Of the 25 compounds evaluated, 24 showed antiviral effects against influenza virus A, 11 showed activity against respiratory syncytial virus, six showed activity against measles virus, and 23 were considered effective against HIV-1 at a lower concentration than that producing cytotoxicity. Four polyoxotungstates which had potent inhibitory effects were examined for inhibitory effects against additional ortho- and paramyxoviruses, and proved to have a broad spectrum of antimyxoviral activity. HS-058, the Keggin sandwich compound K10Fe4(H2O)2(PW9O34)2·nH2O, was inhibitory against influenza viruses A and B, respiratory syncytial virus, measles virus, and parainfluenza virus 2, with median effective concentrations of 1.4, 21.8, 7.4, 0.8 and 0.32 μ,M, respectively. However, HS-058 had no effect on parainfluenza virus 3 or mumps virus. The median cytotoxic concentration of HS-058 for Madin-Darby canine kidney (MDCK) and HEp-2 cells was more than 200 μM and that for HMV-2 and Vero cells was about 50 μM. When HS-058 was added at different times after influenza A and respiratory syncytial virus infection, it inhibited binding of the latter but not of the former to cells. However, at higher concentrations, HS-054 and HS-058 inhibited haemolysis of chick erythrocytes by influenza virus and syncytium formation involving respiratory syncytial virus-infected cells and uninfected cells. Four times the median effective antiviral concentration of HS-058 completely inhibited the growth of influenza virus A in MDCK cells when compound was added before virus adsorption. Furthermore, when HS-058 was added after virus adsorption, it inhibited the yield of virus in MDCK cells infected at low but not at high multiplicity of infection.

Antiviral activity of NMSO3 against adenovirus in vitro

NMSO3, a sulfated sialyl lipid, was evaluated for its efficacy against adenovirus (AdV) in vitro. The median effective concentration (50% effective concentration, EC(50)) of NMSO3 against replication of AdV type 2 (AdV2), type 4 (AdV4), type 8 (AdV8) and type 37 (AdV37) was 0.21-0.71 microg/ml in HEp-2 cells and 1.01-1.41 microg/ml in MKN-28 cells. The EC(50) values of NMSO3 were lower than those of HPMPC and ddC, which were also evaluated. NMSO3 exhibited minimal cytotoxicity against HEp-2 cells and MKN-28 cells, both for which the median cytotoxic concentration (50% cytotoxic concentration, CC(50)) was more than 1000 microg/ml. NMSO3 was the most potent and selective anti-AdV compound of those examined. NMSO3 inhibited AdV infection of HEp-2 cells only when present during the virus adsorption period. A virus binding assay using radiolabeled AdV4 revealed that NMSO3 inhibited viral binding to the HEp-2 cells. NMSO3 itself bound to the virus particles, but not to the HEp-2 cell membrane. Thus, the mechanism of anti-AdV activity by NMSO3 involves inhibition of virus adsorption to cells by NMSO3 binding to viral particles.

High-Dose Intravenous Ribavirin Therapy for Subacute Sclerosing Panencephalitis

ABSTRACT Two patients with subacute sclerosing panencephalitis (SSPE) were treated safely and effectively with high doses of intravenous ribavirin combined with intraventricular alpha interferon. The ribavirin concentrations maintained in the serum and cerebrospinal fluid were higher than those which inhibit SSPE virus replication in vitro and in vivo.

Pharmacokinetics and Effects of Ribavirin following Intraventricular Administration for Treatment of Subacute Sclerosing Panencephalitis

ABSTRACT Ribavirin is a broad-spectrum antiviral drug with inhibitory activity against many RNA viruses, including measles virus. Five patients with subacute sclerosing panencephalitis (SSPE) were treated with ribavirin by intraventricular administration. Although there were transient side effects attributed to ribavirin, such as drowsiness, headache, lip and gingival swelling, and conjunctival hyperemia, intraventricular ribavirin therapy was generally safe and well tolerated. The cerebrospinal fluid (CSF) ribavirin concentration decreased, as described by a monoexponential function, after a single intraventricular dose. There was considerable interindividual variability, however, in the peak level and half-life. We aimed to adjust the individual dose and frequency of intraventricular administration based on the peak level and half-life of ribavirin in the CSF in order to maintain the CSF ribavirin concentration at the target level. Clinical effectiveness (significant neurologic improvement and/or a significant decrease in titers of hemagglutination inhibition antibodies against measles virus in CSF) was observed for four of five patients. For these four patients, CSF ribavirin concentrations were maintained at a level at which SSPE virus replication was almost completely inhibited in vitro and in vivo, whereas the concentration was lower in the patient without clinical improvement. These results suggest that intraventricular administration of ribavirin is effective against SSPE if the CSF ribavirin concentration is maintained at a high level. Intraventricular ribavirin therapy should be pursued further for its potential use for patients with SSPE and might be applied in the treatment of patients with encephalitis caused by other RNA viruses.

Tumor necrosis factor antagonizes inhibitory effect of azidothymidine on human immunodeficiency virus (HIV) replication in vitro

Tumor necrosis factor alpha (TNF-alpha) completely reverses the activity of azidothymidine (AZT) against human immunodeficiency virus type 1 (HIV-1) in MOLT-4 cell cultures. The 50% effective concentration of AZT, required to protect MOLT-4 cells against the cytopathic effect of HIV-1, increased from 5.8 nM in the absence of TNF-alpha to greater than 125 microM in the presence of TNF-alpha (100 U/ml). TNF-alpha also antagonized the anti-HIV-1 activity of dideoxycytidine but did not markedly affect the anti-HIV-1 activity of dextran sulfate. The intracellular phosphorylation pattern of AZT was not changed upon the presence of TNF-alpha.