Inhibitory effect of alpha-methyl-1-adamantane methylamine hydrochloride (rimantadine) on RNA-dependent RNA polymerase induction in culture of cells, infected with influenza virus]

An anti-influenza preparation, rimantadine (alpha-methyl-1-adamantane methylamine hydrochloride) at concentrations of 10--25 mkg/ml depresses the RNA-dependent RNA polymerase induction in a culture of cells infected with influenza virus (fowl plague virus). The inhibitory effect is also observed 2 hours following cell infection. In vitro studies have demonstrated that rimantadine has no effect on the activity of virus-induced RNA-dependent RNA polymerase, as well as on that of RNA-dependent RNA polymerase associated with virus particles.     

Chemotherapy and vaccination: a possible strategy for the control of highly virulent influenza virus.

The influenza A virus [A/Chicken/Pennsylvania/1370/83 (H5N2)] that caused up to 80% mortality among chickens provided a model system for testing the efficacy of chemotherapeutic agents against highly virulent influenza virus. Amantadine and rimantadine administered in drinking water were efficacious both prophylactically and therapeutically. However, under conditions simulating natural transmission of virus, amantadine- and rimantadine-resistant viruses arose and were transmitted to other birds in contact with the infected chickens, causing mortality. Simultaneous administration of inactivated H5N2 vaccine and amantadine provided protection. Thus, chemotherapy may be useful in the treatment of a highly pathogenic influenza virus outbreak in humans or other animals when used in combination with vaccine.     

奥司他韦、利巴韦林和盐酸金刚乙胺对甲型H1N1流感病毒的抑制作用

目的细胞水平测试奥司他韦、利巴韦林和盐酸金刚乙胺对甲型流感H1N1病毒的抑制或杀伤作用。方法通过在MDCK细胞系和甲型H1N1病毒株间建立药物剂量-效应关系确定导致细胞死亡的效力与抑制病毒复制的效力的比值（治疗指数）,测试药物的抗病毒效果。结果奥司他韦、利巴韦林和盐酸金刚乙胺对MDCK细胞的半数中毒浓度分别为（1134.7±186.8）μg/mL、（742.0±76.9）μg/mL、（94.6±1.9）μg/mL,对甲型H1N1病毒的治疗指数（TI）分别为71.19、24.9和3.12。结论奥司他韦对甲型H1N1病毒抑制作用最强,利巴韦林其次,盐酸金刚乙胺对甲型H1N1病毒抑制效果较弱。     

Antioxidant properties of rimantadine in influenza virus infected mice and in some model systems

Influenza virus infection is associated with development of oxidative stress in lung and blood plasma, viz. increase of primary and secondary lipid peroxidation products. It was established that rimantadine treatment led to a decrease of the products of lipid peroxidation in tissues of mice experimentally infected with influenza virus A/Aichi/2/68 (H3N2). The effect is strongest in blood plasma (a decrease of about 50%) and weaker in the lung (about 20%). To elucidate the mechanism of this action of rimantadine, experiments were carried out with some model systems. The capability of rimantadine to scavenge superoxide radicals (scavenging properties) was studied in a system of xanthine-xanthine oxidase to generate superoxide. The amount of superoxide was measured spectrophotometrically by the NBT-test and chemiluminesce. Rimantadine does not show scavenging properties and its antioxidant effect observed in vivo, is not a result of its direct action on the processes of lipid peroxidation and/or interaction with antioxidant enzymes. The antioxidant properties of rimantadine were investigated by measurement of induced lipid peroxidation in a Fe2+ and (Fe2+ - EDTA) system with an egg liposomal suspension. Our findings with model systems do not prove an antioxidant or prooxidant effect of the drug on the processes of lipid peroxidation. Apparently, the observed antioxidant effect of rimantadine in vivo is not connected directly with free radical processes in the organism.     

Comparison of in vitro and in vivo methods of evaluation of the efficacy of influenza virus hemagglutinin inhibitors

One of the problem in the selection of the most effective antiviral preparations with a broad spectrum of antiviral protective activity, is the "continuity" of assays of different level of complexity so, that the most effective antiviral therapeutic, selected by in vitro assays would be the most effective in vivo. Comparative study of the efficacy of the influenza virus inhibitor in the assays of inhibition of virus binding with fetuin, inhibition of infectious focus forming units in MDCK cells, inhibition of virus yield in infected MDCK cells, and inhibition of influenza virus infectivity in mice infected by viral aerosol are presented. The value of 50% inhibiting concentration IC50 for the pare "influenza virus strain A/NIB/23/89-MA-inhibitor tetra-Aca6-6'SLN" corresponded to 6-10 microM and was invariant for three different tests--in vitro assay of inhibition of virus binding with fetuin, inhibition of yield in infected MDCK cell culture, and inhibition of virus infectivity in mice, but not for the assay of inhibition of infectious focus forming units in cell culture.     

Kinetics of influenza A virus infection in humans

Currently, little is known about the viral kinetics of influenza A during infection within an individual. We utilize a series of mathematical models of increasing complexity, which incorporate target cell limitation and the innate interferon response, to examine influenza A virus kinetics in the upper respiratory tracts of experimentally infected adults. The models were fit to data from an experimental H1N1 influenza A/Hong Kong/123/77 infection and suggest that it is important to include the eclipse phase of the viral life cycle in viral dynamic models. Doing so, we estimate that after a delay of approximately 6 h, infected cells begin producing influenza virus and continue to do so for approximately 5 h. The average lifetime of infected cells is approximately 11 h, and the half-life of free infectious virus is approximately 3 h. We calculated the basic reproductive number, R(0), which indicated that a single infected cell could produce approximately 22 new productive infections. This suggests that antiviral treatments have a large hurdle to overcome in moderating symptoms and limiting infectiousness and that treatment has to be initiated as early as possible. For about 50% of patients, the curve of viral titer versus time has two peaks. This bimodal behavior can be explained by incorporating the antiviral effects of interferon into the model. Our model also compared well to an additional data set on viral titer after experimental infection and treatment with the neuraminidase inhibitor zanamivir, which suggests that such models may prove useful in estimating the efficacies of different antiviral therapies for influenza A infection.     

Selective response of gamma delta T-cell hybridomas to orthomyxovirus-infected cells.

A gamma delta T-cell hybridoma established from influenza virus-infected mice responded to a reproducible way when cultured with influenza virus-infected stimulators. Subclones of this line responded to cells infected with influenza viruses A/PR/8/34 (H1N1), X-31 (H3N2), and B/HK/8/73 but not to cells infected with vaccinia virus or Sendai virus. This spectrum of response to both type A and type B orthomyxoviruses has never been recognized for the alpha beta T-cell receptor-positive subsets. There was no response to cells infected with a panel of recombinant vaccinia viruses expressing all individual influenza virus proteins, and so it is unlikely that the stimulating antigen is of viral origin. The alternative is that the antigen is a cellular molecule induced in influenza virus-infected cells. Infectious virus was required for stimulation, and immunofluorescence studies showed increased expression of heat shock protein 60 (Hsp60) in influenza virus- but not Sendai virus- or vaccinia virus-infected cells. Both the hybridoma generated from influenza virus-infected mice and an established hybridoma which uses the same gamma delta T-cell receptor combination responded to recombinant Hsp60. Furthermore, the Hsp60-reactive hybridoma, which was obtained from an uninfected mouse, also responded to influenza virus-infected cells, indicating that Hsp60 may indeed be the target antigen.     

Heterocyclic rimantadine analogues with antiviral activity

2-(1-Adamantyl)-2-methyl-pyrrolidines 3 and 4, 2-(1-adamantyl)-2-methyl-azetidines 5 and 6, and 2-(1-adamantyl)-2-methyl-aziridines 7 and 8 were synthesized and tested for their antiviral activity against influenza A. Parent molecules 3, 5, and 7 contain the alpha-methyl-1-adamantan-methanamine 2 pharmacophoric moiety (rimantadine). The ring size effect on anti-influenza A activity was investigated. Pyrrolidine 3 was the most potent anti-influenza virus A compound, 9-fold more potent than rimantadine 2, 27-fold more potent than amantadine 1, and 22-fold more potent than ribavirin. Azetidines 5 and 6 were both markedly active against influenza A H2N2 virus, 10- to 20-fold more potent than amantadine. Aziridine 7 was almost devoid of any activity against H2N2 virus but exhibited borderline activity against H3N2 influenza A strain. Thus, it appears that changing the five-, to four- to a three-membered ring results in a drop of activity against influenza A virus.     

Virus specificity of human influenza virus-immune cytotoxic T cells.

The virus specificity of human in vitro cytotoxic T cell responses to influenza virus was studied with the use of peripheral blood mononuclear leukocytes from normal adult volunteers. Previous natural exposure of these donors to a variety of type A influenza viruses was documented by HI antibody titers. Cells sensitized in vitro with A/HK or A/PR8 were cytotoxic for autologous target cells infected with A/HK, A/PR8, or A/JAP 305 type A influenza viruses, but not for B/HK-infected or uninfected cells. B/HK-sensitized effector cells lysed target cells infected with B/HK but not targets infected with type A viruses. A/HK- and A/PR8-immune effector populations were shown to recognize cross-reactive antigens on A/HK- and A/PR8-infected target cells by cold target competition. Influenza-immune effector cells were cytotoxic for virus-infected autologous targets but much less so for virus-infected allogeneic targets. This self-restriction suggested that the cytotoxicity was largely T cell-mediated and was confirmed by cell separation analysis. Thus, the human secondary cytotoxic T cell response in vitro to influenza viruses is predominantly directed against cross-reactive determinants on cells infected with serologically distinct type A influenza viruses.     

Immunologic recognition of influenza virus-infected cells. I. Generation of a virus-strain specific and a cross-reactive subpopulation of cytotoxic T cells in the response to type A influenza viruses of different subtypes.

Parenteral immunization of mice with a given strain of type A influenza virus generates two subpopulations of cytotoxic T cells in the in vivo primary response. One subpopulation is specific for the immunizing virus; the other subpopulation cross-reacts with target cells infected with type A influenza virus of a different subtype. Both subpopulations are specific for target cells infected with type A influenza virus and optimally lyse only infected targets which are syngeneic at the H-2 gene locus. In vitro stimulation of previously primed spleen cells with cells infected with homologous virus generates both subpopulations in the secondary cytotoxic response. However, in vitro stimulation of primed cells with cells infected with heterologous type A virus of a different subtype specifically selects for the cross-reactive T-cell population. These results are discussed in terms of current models for T-cell recognition of virus-infected cells and possible mechanisms for cross-reaction between type A influenza viruses of different subtypes at the level of cytotoxic T cells.     

Conformational changes induced by a single amino acid substitution in the trans-membrane domain of Vpu: implications for HIV-1 susceptibility to channel blocking drugs

The channel-forming trans-membrane domain of Vpu (Vpu TM) from HIV-1 is known to enhance virion release from the infected cells and is a potential target for ion-channel blockers. The substitution of alanine at position 18 by a histidine (A18H) has been shown to render HIV-1 infections susceptible to rimantadine, a channel blocker of M2 protein from the influenza virus. In order to describe the influence of the mutation on the structure and rimantadine susceptibility of Vpu, we determined the structure of A18H Vpu TM, and compared it to those of wild-type Vpu TM and M2 TM. Both isotropic and orientationally dependent NMR frequencies of the backbone amide resonance of His18 were perturbed by rimantadine, and those of Ile15 and Trp22 were also affected, suggesting that His18 is the key residue for rimantadine binding and that residues located on the same face of the TM helix are also involved. A18H Vpu TM has an ideal, straight alpha-helix spanning residues 6-27 with an average tilt angle of 41 degrees in C14 phospholipid bicelles, indicating that the tilt angle is increased by 11 degrees compared to that of wild-type Vpu TM. The longer helix formed by the A18H mutation has a larger tilt angle to compensate for the hydrophobic mismatch with the length of the phospholipids in the bilayer. These results demonstrate that the local change of the primary structure plays an important role in secondary and tertiary structures of Vpu TM in lipid bilayers and affects its ability to interact with channel blockers.     

Mathematical model of influenza A virus production in large-scale microcarrier culture

A mathematical model that describes the replication of influenza A virus in animal cells in large-scale microcarrier culture is presented. The virus is produced in a two-step process, which begins with the growth of adherent Madin-Darby canine kidney (MDCK) cells. After several washing steps serum-free virus maintenance medium is added, and the cells are infected with equine influenza virus (A/Equi 2 (H3N8), Newmarket 1/93). A time-delayed model is considered that has three state variables: the number of uninfected cells, infected cells, and free virus particles. It is assumed that uninfected cells adsorb the virus added at the time of infection. The infection rate is proportional to the number of uninfected cells and free virions. Depending on multiplicity of infection (MOI), not necessarily all cells are infected by this first step leading to the production of free virions. Newly produced viruses can infect the remaining uninfected cells in a chain reaction. To follow the time course of virus replication, infected cells were stained with fluorescent antibodies. Quantitation of influenza viruses by a hemagglutination assay (HA) enabled the estimation of the total number of new virions produced, which is relevant for the production of inactivated influenza vaccines. It takes about 4-6 h before visibly infected cells can be identified on the microcarriers followed by a strong increase in HA titers after 15-16 h in the medium. Maximum virus yield Vmax was about 1x10(10) virions/mL (2.4 log HA units/100 microL), which corresponds to a burst size ratio of about 18,755 virus particles produced per cell. The model tracks the time course of uninfected and infected cells as well as virus production. It suggests that small variations (<10%) in initial values and specific rates do not have a significant influence on Vmax. The main parameters relevant for the optimization of virus antigen yields are specific virus replication rate and specific cell death rate due to infection. Simulation studies indicate that a mathematical model that neglects the delay between virus infection and the release of new virions gives similar results with respect to overall virus dynamics compared with a time delayed model.     

A mutation on influenza C virus M1 protein affects virion morphology by altering the membrane affinity of the protein

Reverse genetics has been documented for influenza A, B, and Thogoto viruses belonging to the family Orthomyxoviridae. We report here the reverse genetics of influenza C virus, another member of this family. The seven viral RNA (vRNA) segments of C/Ann Arbor/1/50 were expressed in 293T cells from cloned cDNAs, together with nine influenza C virus proteins. At 48 h posttransfection, the infectious titer of the culture supernatant was determined to be 2.51 x 10(3) 50% egg infectious doses/ml, which is lower than the number of influenza C virus-like particles (VLPs) (10(6)/ml) generated using the same system. By generating influenza C VLPs containing a given vRNA segment, we showed that each of the vRNA segments was similarly synthesized in the plasmid-transfected cells but that some segments were less efficiently incorporated into the VLPs. This finding leads us to speculate that the differences in incorporation efficiency into VLPs between segments might be a reason for the inefficient production of infectious viruses. Second, we generated a mutant recombinant virus, rMG96A, which possesses an Ala-->Thr mutation at residue 24 of the M1 protein, a substitution demonstrated to be involved in the morphology (filamentous or spherical) of the influenza C VLPs. As expected, rMG96A exhibited a spherical morphology, whereas recombinant wild-type of C/Ann Arbor/1/50, rWT, exhibited a mainly filamentous morphology. Membrane flotation analysis of the cells infected with rWT or rMG96A revealed a difference in the ratio of membrane-associated M1 proteins, suggesting that the affinity of M1 protein to the cell membrane is a determinant for virion morphology.     

Insights from investigating the interactions of adamantane-based drugs with the M2 proton channel from the H1N1 swine virus

The M2 proton channel is one of indispensable components for the influenza A virus that plays a vital role in its life cycle and hence is an important target for drug design against the virus. In view of this, the three-dimensional structure of the H1N1-M2 channel was developed based on the primary sequence taken from a patient recently infected by the H1N1 (swine flu) virus. With an explicit water-membrane environment, molecular docking studies were performed for amantadine and rimantadine, the two commercial drugs generally used to treat influenza A infection. It was found that their binding affinity to the H1N1-M2 channel is significantly lower than that to the H5N1-M2 channel, fully consistent with the recent report that the H1N1 swine virus was resistant to the two drugs. The findings and the relevant analysis reported here might provide useful structural insights for developing effective drugs against the new swine flu virus.     

Autocrine type I IFN and contact with endothelium promote the presentation of influenza A virus by monocyte-derived APC

Purified monocytes infected with influenza A virus do not become mature dendritic cells (DCs) and they present viral peptides poorly to autologous memory T cells. In this study, we investigated whether influenza A-infected monocytes matured to DCs with a high capacity to stimulate T cells when they were infected with influenza A virus in a model tissue setting wherein they were cocultured with endothelium grown on a type I collagen matrix. Intercellular interactions with endothelium strongly promoted the Ag-presenting capacity of monocyte-derived cells infected with influenza A virus, and the heterologous coculture system also enhanced production of IFN-alpha by monocytes in the absence of plasmacytoid cells. Production of IFN-alpha in the presence of endothelium correlated with monocyte differentiation to mature DCs and their ability to stimulate proliferation and IFN-gamma production by autologous T cells. Monocyte-derived cells that developed into migratory DCs promoted proliferation of influenza A virus-specific CD4(+) and CD8(+) cells, whereas those that developed into macrophages promoted proliferation of CD8(+) T cells only. This onset of APC activity could be partially blocked with Ab to the IFN-alphabeta receptor when monocytes were infected with UV-treated virus, but neutralizing this pathway was inconsequential when monocytes were infected with live virus. Thus, type I IFN and direct contact with endothelium promote development of influenza A virus-presenting activity in monocyte-derived cells in a setting in which this differentiation does not depend on plasmacytoid cells. However, when infected with live influenza virus, the role of type I IFN in mediating differentiation and Ag-presenting capacity is expendable, apparently due to other mechanisms of virus-mediated activation.     

Infection of cultured human airway epithelial cells by influenza A virus

The lack of an adequate in vitro model has hampered study of the cellular basis by which influenza A virus causes disease in the human airway. We report in vitro infection of human airway epithelial cells by influenza A virus. Fetal and adult human tracheal and bronchial epithelial cells cultured from explants and SV40 transformed adult human tracheal epithelial cells were exposed to a recently isolated strain of influenza A virus (H1N1) and a laboratory passaged strain (WSN) of influenza A virus at similar multiplicity of infection. All cultures derived from explants showed hemadsorption (approximately 30% of the cells) with the H1N1 virus. No hemadsorption was detected with the WSN virus. One of two transformed cell lines showed a 5-10% hemadsorption to cells after H1N1 exposure and none following exposure to WSN. Immunofluorescent staining for influenza A-specific antigens in virus-exposed, explant-derived cells indicated viral infection and replication in these cells. Hemagglutinating material in the growth medium of infected, explant-derived cell lines, increased as a function of time, indicating the production of virus proteins. Exposure of rhesus monkey kidney cells and new human tracheal epithelial cultures to supernatant from these cells resulted in hemadsorption, indicating the presence of infectious virus in the supernatant. Light microscopic examination of virally infected bronchial epithelial cells demonstrated that the common types of cytopathic changes were rarely seen while cell proliferation continued over time. The data indicate that influenza A virus can infect, replicate, and produce infectious virus in cultured human tracheal and bronchial epithelial cells.     

Anti-Influenza A Virus Effect of Hypericum perforatum L. Extract

To study the antiviral effect of Hypericum perforatum L. extract (HPE) on influenza A virus (IAV) (H1N1) in vitro and in vivo. Cytopathic effect (CPE) and neutral red (NR) dye uptake were used to examine the antiviral effect of HPE on Madin Darby Canine Kidney (MDCK) cells which were infected with IAV in vitro. HPE was effective against influenza A virus (IAV) in vitro, with a 50% effective concentration (EC50) of 40 μg/mL. The mean 50% cytotoxic concentration (CC50) in the MDCK used in these experiments was 1.5 mg/mL. Ribavirin was run in parallel with EC50 values of 5.0 μg/mL; the mean CC50 for ribavirin was 520 μg/mL. Oral gavage administrations of HPE or ribavirin to mice infected with the IAV were highly effective in preventing death, slowing the decline of arterial oxygen saturation, inhibiting lung consolidation and reducing lung virus titers. The minimum effective dose of HPE in these studies was 31.25 mg/kg/day, which was administered twice daily for 5 d beginning 4 h prior to virus exposure. Below a dosage of 2000 mg/kg/day, almost all treated mice survived, which suggests that HPE is of low toxicity. Ribavirin's minimum effective dose was 40 mg/kg/day with the LD50 determined to be 200 mg/kg/day. Delay of the initiation of either HPE or ribavirin therapy, using approximately 1/3 LD50 dose each time, could still be protective as late as 48 h after exposure to the IAV. While both agents appeared to have similar efficacy against IAV infections, HPE was considered to be less toxic and may warrant further evaluation as a possible therapy for influenza.     

Virions and intracellular nucleocapsids produced during mixed heterotypic influenza infection of MDCK cells.

Phenotypically mixed virus yields, obtained by coinfection of MDCK cells with influenza A/WSN/33 and B/Lee/40 viruses, contained both A/WSN/33 and B/Lee/40 NP proteins, as revealed by polyacrylamide gel electrophoresis of the purified 14C-amino acids-labeled virus. Virions were lysed with 0.6 M KCl-Triton X-100 buffer, and nucleocapsids were immunoprecipitated with antibodies against NP protein of influenza A virus. Polyacrylamide gel electrophoresis of the immunoprecipitate revealed NP protein of A/WSN/33 but not of B/Lee/40 virus. However, in similar experiments with the lysates of doubly infected cells, the band of B/Lee/40 NP protein was revealed in the polyacrylamide gel electrophoresis patterns of the immunoprecipitates. In an attempt to analyze the RNA content of the immune complexes, we absorbed the lysates of doubly infected [3H]uridine-labeled cells with protein A-containing Staphylococcus aureus covered with antibodies against the NP protein of influenza A virus. RNA extracted from the immune complexes contained genomic RNA segments of both A/WSN/33 and B/Lee/40 viruses. In control samples containing an artificial mixture of cell lysates separately infected with each virus, the analysis revealed homologous components (i.e., A/WSN/33 NP protein or RNA segments) in the immune complexes. The results suggest the presence of phenotypically mixed nucleocapsids in the cells doubly infected with influenza A and B viruses; in the course of the virion formation, the nucleocapsids lacking the heterologous NP protein are selected.     

Design of an Efficient Inhibitor for the Influenza A Virus M2 Ion Channel

Influenza A virus is capable of rapidly infecting large human populations, warranting the development of novel drugs to efficiently inhibit virus replication. A transmembrane ion channel formed by the M2 protein plays an important role in influenza virus replication. A reasonable approach to designing an effective antivirus drug is constructing a molecule that binds in the M2 transmembrane proton channel, blocks H+ proton diffusion through the channel, and thus the influenza A virus cycle. The known anti-influenza drugs amantadine and rimantadine have a weak effect on influenza A virus replication. A new class of positively charged molecules, diazabicyclooctane derivatives with a constant charge of +2, was proposed to block proton diffusion through the M2 ion channel. Molecular dynamics simulations were performed to study the temperature fluctuations in the M2 structure, and ionization states of histidine residues were established at physiological pH values. Two types of diazabicyclooctane derivatives were analyzed for binding with the M2 ion channel. An optimal structure was determined for a blocker to most efficiently bind with the M2 ion channel and block proton diffusion. The new molecule is advantageous over amantadine and rimantadine in having a positive charge of +2, which creates a positive electrostatic potential barrier to proton transport through the M2 ion channel in addition to a steric barrier.