Rimantadine but not amantadine protects Fischer rat embryo cells from transformation induced by 3-methylcholanthrene or benzo(a)pyrene.

The antiviral drugs amantadine hydrochloride and rimantadine hydrochloride were tested as to their oncogenic potential using a serial line of Fischer rat embryo cells that previously had been shown to be an accurate indicator of chemicals known to be oncogenic in animal studies. Neither compound was found to have transforming activity. At slightly toxic levels, rimanbadine hydrochloride, but not amantadine hydrochloride, protected the same cell line from the transformation induced by the polycyclic hydrocarbons 3-methylcholanthrene and benzo(a)pyrene.     

Antiviral Activity of Polyacrylic and Polymethacrylic Acids: I. Mode of Action in Vitro.

Polyacrylic acid (PAA) and polymethacrylic acid (PMAA) were investigated for their antiviral properties in tissue culture. Compared to other related polyanions, as dextran sulfate, polystyrene sulfonate, polyvinyl sulfate, and polyphloroglucinol phosphate, PAA and PMAA were found to be significantly more antivirally active and less cytotoxic. PMAA added 24 hr prior to virus inoculation inhibited viral growth most efficiently but it was still effective when added 3 hr after infection. Neither a direct irreversible action on the virus nor inhibition of virus penetration into the cell could explain the antiviral activity of PMAA. PMAA inhibited the adsorption of the virus to the host cell and suppressed the one-cycle viral synthesis in tissue cultures inoculated with infectious RNA.     

Studies on the Mechanism of Interferon Action

Interferon does not inactivate viruses or viral RNA. Virus growth is inhibited in interferon-treated cells, but apart from conferring resistance to virus growth, no other effect of interferon on cells has been definitely shown to take place. Interferon binds to cells even in the cold, but a period of incubation at 37°C is required for development of antiviral activity. Cytoplasmic uptake of interferon has not been unequivocally demonstrated. Studies with antimetabolites indicate that the antiviral action of interferon requires host RNA and protein synthesis. Experiments with 2-mercapto-1(β-4-pyridethyl) benzimidazole (MPB) suggest that an additional step is required between the binding and the synthesis of macromolecules. Interferon does not affect the adsorption, penetration, or uncoating of RNA or DNA viruses, but viral RNA synthesis is inhibited in cells infected with RNA viruses. The main action of interferon appears to be the inhibition of the translation of virus genetic information probably by inhibiting the initiation of virus protein synthesis.     

Rimantadine but not amantadine protects fischer rat embryo cells from transformation induced by 3-methylcholanthrene or benzo(a)pyrene

Summary The antiviral drugs amantadine hydrochloride and rimantadine hydrochloride were tested as to their oncogenic potential using a serial line of Fischer rat embryo cells that previously had been shown to be an accurate indicator of chemicals known to be oncogenic in animal studies. Neither compound was found to have transforming activity. At slightly toxic levels, rimantadine hydrochloride, but not amantadine hydrochloride, protected the same cell line from the transformation induced by the polycyclic hydrocarbons 3-methylcholanthrene and benzo(a)pyrene. This work was supported by Contract N01-CP-43240 within The Virus Cancer Program of the National Cancer Institute.     

A Dimer Is the Minimal Proton-Conducting Unit of the Influenza A Virus M2 Channel

When influenza A virus infects host cells, its integral matrix protein M2 forms a proton-selective channel in the viral envelope. Although X-ray crystallography and NMR studies using fragment peptides have suggested that M2 stably forms a tetrameric channel irrespective of pH, the oligomeric states of the full-length protein in the living cells have not yet been assessed directly. In the present study, we utilized recently developed stoichiometric analytical methods based on fluorescence resonance energy transfer using coiled-coil labeling technique and spectral imaging, and we examined the relationship between the oligomeric states of full-length M2 and its channel activities in living cells. In contrast to previous models, M2 formed proton-conducting dimers at neutral pH and these dimers were converted to tetramers at acidic pH. The antiviral drug amantadine hydrochloride inhibited both tetramerization and channel activity. The removal of cholesterol resulted in a significant decrease in the activity of the dimer. These results indicate that the minimum functional unit of the M2 protein is a dimer, which forms a complex with cholesterol for its function.     

Comparative analysis of poxvirus orthologues of the vaccinia virus E3 protein: modulation of protein kinase R activity, cytokine responses, and virus pathogenicity

Poxviruses are important human and animal pathogens that have evolved elaborate strategies for antagonizing host innate and adaptive immunity. The E3 protein of vaccinia virus, the prototypic member of the orthopoxviruses, functions as an inhibitor of innate immune signaling and is essential for vaccinia virus replication in vivo and in many human cell culture systems. However, the function of orthologues of E3 expressed by poxviruses of other genera with different host specificity remains largely unknown. In the present study, we characterized the E3 orthologues from sheeppox virus, yaba monkey tumor virus, swinepox virus, and myxoma virus for their ability to modulate protein kinase R (PKR) function, cytokine responses and virus pathogenicity. We found that the E3 orthologues of myxoma virus and swinepox virus could suppress PKR activation and interferon (IFN)-induced antiviral activities and restore the host range function of E3 in HeLa cells. In contrast, the E3 orthologues from sheeppox virus and yaba monkey tumor virus were unable to inhibit PKR activation. While the sheeppox orthologue was unable to restore the host range function of E3, the yaba monkey tumor virus orthologue partially restored E3-deficient vaccinia virus replication in HeLa cells, correlated with its ability to suppress IFN-induced antiviral activities. Moreover, poxvirus E3 orthologues show varying ability to inhibit the induction of antiviral and proinflammatory cytokines. Despite these in vitro results, none of the E3 orthologues tested was capable of restoring pathogenicity to E3-deficient vaccinia virus in vivo.     

盐酸阿比朵尔抗柯萨奇病毒B3的实验研究

为观察盐酸阿比朵尔对柯萨奇病毒的抑制作用,本实验以利巴韦林为阳性对照药物,采用细胞培养技术、细胞病变效应(CPE)抑制法、活细胞染色计数法(MTT)和培养上清中病毒滴度测定观察盐酸阿比朵尔抗柯萨奇病毒的作用。结果表明盐酸阿比朵尔的半数中毒浓度(TD50)为896.54μg/mL,药物抗病毒生物合成组、药物直接作用组、药物抗病毒吸附2h组、药物抗病毒有吸附8h组的病毒抑制率分别能达到74.48%、45.68%、28.90%和48.27%,在抗生物合成组,盐酸阿比朵尔能明显抑制柯萨奇病毒所致的CPE效应,降低培养上清中的病毒滴度,病毒抑制率随药物浓度增加而增高,存在量效关系。这提示盐酸阿比朵尔在细胞内对柯萨奇病毒有一定的抑制作用。     

The vaccinia virus soluble alpha/beta interferon (IFN) receptor binds to the cell surface and protects cells from the antiviral effects of IFN

Poxviruses encode a broad range of proteins that interfere with host immune functions, such as soluble versions of receptors for the cytokines tumor necrosis factor, interleukin-1 beta, gamma interferon (IFN-gamma), IFN-alpha/beta, and chemokines. These virus-encoded cytokine receptors have a profound effect on virus pathogenesis and enable the study of the role of cytokines in virus infections. The vaccinia virus (VV) Western Reserve gene B18R encodes a secreted protein with 3 immunoglobulin domains that functions as a soluble receptor for IFN-alpha/beta. We have found that after secretion B18R binds to both uninfected and infected cells. The B18R protein present at the cell surface maintains the properties of the soluble receptor, binding IFN-alpha/beta with high affinity and with broad species specificity, and protects cells from the antiviral state induced by IFN-alpha/beta. VV strain Wyeth expressed a truncated B18R protein lacking the C-terminal immunoglobulin domain. This protein binds IFN with lower affinity and retains its ability to bind to cells, indicating that the C-terminal region of B18R contributes to IFN binding. The replication of a VV B18R deletion mutant in tissue culture was restricted in the presence of IFN-alpha, whereas the wild-type virus replicated normally. Binding of soluble recombinant B18R to cells protected the cultures from IFN and allowed VV replication. This represents a novel strategy of virus immune evasion in which secreted IFN-alpha/beta receptors not only bind the soluble cytokine but also bind to uninfected cells and protect them from the antiviral effects of IFN-alpha/beta, maintaining the cells' susceptibility to virus infections. The adaptation of this soluble receptor to block IFN-alpha/beta activity locally will help VV to replicate in the host and spread in tissues. This emphasizes the importance of local effects of IFN-alpha/beta against virus infections.     

Antiviral Effect of Cyclic Amines on a 1969 Isolate of A2 Influenza

Cyclooctylamine and amantadine inhibit the growth of 1969 isolates of A(2) influenza virus to a significant degree. There was slightly more inhibition of the virus by the cyclooctylamine (COA) than the amantadine; however, the dose of COA used was greater than the dose of amantadine. There was no significant difference between flasks treated 3 or 4 hr and those treated 2 hr. However, there was a curious relationship of more plaques in the flasks exposed to the two drugs for the longer intervals. Other experiments done with slight modifications in technique support the antiviral effect demonstrated in this experiment when the cell system is pretreated prior to virus infection. In two experiments, pretreating the cells for 2 hr with COA at 100 mug/ml but removing the drug solution and washing the cells prior to virus inoculation revealed no differences in plaque counts between controls and treated cells. This would indicate that the antiviral effect required the presence of the drug during the early stages of penetration of the cells by the virus particles.     

Live Cell Analysis and Mathematical Modeling Identify Determinants of Attenuation of Dengue Virus 2’-O-Methylation Mutant

Dengue virus (DENV) is the most common mosquito-transmitted virus infecting ~390 million people worldwide. In spite of this high medical relevance, neither a vaccine nor antiviral therapy is currently available. DENV elicits a strong interferon (IFN) response in infected cells, but at the same time actively counteracts IFN production and signaling. Although the kinetics of activation of this innate antiviral defense and the timing of viral counteraction critically determine the magnitude of infection and thus disease, quantitative and kinetic analyses are lacking and it remains poorly understood how DENV spreads in IFN-competent cell systems. To dissect the dynamics of replication versus antiviral defense at the single cell level, we generated a fully viable reporter DENV and host cells with authentic reporters for IFN-stimulated antiviral genes. We find that IFN controls DENV infection in a kinetically determined manner that at the single cell level is highly heterogeneous and stochastic. Even at high-dose, IFN does not fully protect all cells in the culture and, therefore, viral spread occurs even in the face of antiviral protection of naïve cells by IFN. By contrast, a vaccine candidate DENV mutant, which lacks 2’-O-methylation of viral RNA is profoundly attenuated in IFN-competent cells. Through mathematical modeling of time-resolved data and validation experiments we show that the primary determinant for attenuation is the accelerated kinetics of IFN production. This rapid induction triggered by mutant DENV precedes establishment of IFN-resistance in infected cells, thus causing a massive reduction of virus production rate. In contrast, accelerated protection of naïve cells by paracrine IFN action has negligible impact. In conclusion, these results show that attenuation of the 2’-O-methylation DENV mutant is primarily determined by kinetics of autocrine IFN action on infected cells.     

Berbamine hydrochloride potently inhibits SARS-CoV-2 infection by blocking S protein-mediated membrane fusion

COVID-19 caused by SARS-CoV-2 has posed a significant threat to global public health since its outbreak in late 2019. Although there are a few drugs approved for clinical treatment to combat SARS-CoV-2 infection currently, the severity of the ongoing global pandemic still urges the efforts to discover new antiviral compounds. As the viral spike (S) protein plays a key role in mediating virus entry, it becomes a potential target for the design of antiviral drugs against COVID-19. Here, we tested the antiviral activity of berbamine hydrochloride, a bis-benzylisoquinoline alkaloid, against SARS-CoV-2 infection. We found that berbamine hydrochloride could efficiently inhibit SARS-CoV-2 infection in different cell lines. Further experiments showed berbamine hydrochloride inhibits SARS-CoV-2 infection by targeting the viral entry into host cells. Moreover, berbamine hydrochloride and other bis-benzylisoquinoline alkaloids could potently inhibit S-mediated cell-cell fusion. Furthermore, molecular docking results implied that the berbamine hydrochloride could bind to the post fusion core of SARS-CoV-2 S2 subunit. Therefore, berbamine hydrochloride may represent a potential efficient antiviral agent against SARS-CoV-2 infection.     

Characterization of an antiviral agent from primary murine fibroblast cultures: murine tissue culture CVI

We have previously described a class of virus inhibitors which are produced spontaneously by many types of cells in culture and present in a number of physiological fluids. These inhibitors are differentiated from all other known naturally occurring antiviral substances in regard to their (i) lack of species specificity, (ii) broad antiviral activity (iii) absence of high affinity binding by the inhibitor to the virus, (iv) mechanism of the action of the inhibitor is through inhibition of viral attachment, and (v) extreme thermal stability. In this report, we show that this class of inhibitors can be divided into two distinct subclasses. The first category includes the inhibitor spontaneously produced by cells in culture, originally described as contact-blocking viral inhibitor (CVI), and has a polypeptide component associated with its antiviral activity. The second category includes the inhibitors detected in body fluids and tissue extracts and has no essential peptide structure. Further characterization of CVI with respect to molecular size and stability to heat and a number of chemical reagents and enzymes indicate that the antiviral activity of CVI is associated with a large molecule (90s or approximately 4 million daltons), is stable at 100(8)C, and is resistant to the action of RNase, DNase, sulfhydral reagents, protein denaturants, and extraction by organic solvents.     

Host DNA Synthesis-Suppressing Factor in Culture Fluid of Tissue Cultures Infected with Measles Virus

Host DNA synthesis is suppressed by the culture fluid of cell cultures infected with measles virus. This activity in the culture fluid is initiated somewhat later than the growth of infectious virus. Ninety percent of host DNA synthesis in HeLa cells is inhibited by culture fluid of 3-day-old cell cultures of Vero or HeLa cells infected with measles virus. This suppressing activity is not a property of the virion, but is due to nonvirion-associated component which shows none of the activities of measles virus such as hemagglutination, hemolysis, or cell fusion nor does it have the antigenicity of measles virus as tested by complement-fixation or hemagglutination-inhibiting antibody blocking tests. Neutralization of the activity of this component is not attained with the pooled sera of convalescent measles patients. This component has molecular weights of about 45,000, 20,000, and 3,000 and appears to be a heat-stable protein. The production of host DNA suppressing factor (DSF) is blocked by cycloheximide. Neither UV-inactivated nor antiserum-neutralized measles virus produce DSF. Furthermore, such activity of nonvirion-associated component is not detected in the culture fluid of cultures infected with other RNA viruses such as poliovirus, vesicular stomatitis virus, or Sindbis virus.     

Exclusion of superinfecting homologous virus by Sindbis virus-infected Aedes albopictus (mosquito) cells.

The infection of tissue-cultured Aedes albopictus (mosquito) cells by an alphavirus ultimately results in a persistently infected cell population which can be maintained in the laboratory for years. One characteristic of this culture is that it will not support the replication of superinfecting homologous virus. We have previously shown that mosquito cells persistently infected with Sindbis virus produce an antiviral agent which when applied to uninfected mosquito cells suppresses Sindbis virus replication. The exclusion of virus replication in the antiviral-agent-treated cells is similar to the phenomenon of homologous interference described in alphavirus-infected vertebrate cells. In this study we examined the expression of homologous interference in three lines of mosquito cells and compared the expression of homologous interference to the effects of the antiviral activity. The cell lines were found to differ in their ability to express homologous interference, and evidence suggests that the mosquito cells may suppress replication by homologous interference or by the action of the antiviral agent.     

In vitro inhibition of the cytopathic action of herpes simplex virus, type 1, with a natural cytokine complex

The antiviral action of a natural cytokine complex (NCC)--the preparation Superlymph and its peptide antimicrobial fraction (AMF)--in the culture of Vero cells infected with type 1 herpes simplex virus (HSV-1), strain VR-3, was studied. The NCC preparation did not alter the morphology of the cells for 6 days and was not toxic for the culture of Vero cells. The NCC and AMF produced a protective antiviral effect, which was manifested by the inhibition of the cytopathic action (CPA) of the virus. In the presence of the preparation, the CPA of HSV-1 was equal to 10(-4.67) ICPD50, while in the control CPA was equal to 10(-5.60). The fraction containing antimicrobial peptides (protegrins) and isolated from NCC, characterized by the method of mass spectrometry, produced the maximum antiviral effect on the cell strain Vero (10(-4.58) ICPD50). Thus Superlymph, an immunomodulator with antiviral activity, could be regarded as an effective preparation for the treatment of HSV infection. The action of such preparation was aimed at the inhibition of the CPA of the virus and the stimulation of the antiviral protective mechanisms of the cell.     

Cyanovirin-N inhibits hepatitis C virus entry by binding to envelope protein glycans

Inhibition of viruses at the stage of viral entry provides a route for therapeutic intervention. Because of difficulties in propagating hepatitis C virus (HCV) in cell culture, entry inhibitors have not yet been reported for this virus. However, with the development of retroviral particles pseudotyped with HCV envelope glycoproteins (HCVpp) and the recent progress in amplification of HCV in cell culture (HCVcc), studying HCV entry is now possible. In addition, these systems are essential for the identification and the characterization of molecules that block HCV entry. The lectin cyanovirin-N (CV-N) has initially been discovered based on its potent activity against human immunodeficiency virus. Because HCV envelope glycoproteins are highly glycosylated, we sought to determine whether CV-N has an antiviral activity against this virus. CV-N inhibited the infectivity of HCVcc and HCVpp at low nanomolar concentrations. This inhibition is attributed to the interaction of CV-N with HCV envelope glycoproteins. In addition, we showed that the carbohydrate binding property of CV-N is involved in the anti-HCV activity. Finally, CV-N bound to HCV envelope glycoproteins and blocked the interaction between the envelope protein E2 and CD81, a cell surface molecule involved in HCV entry. These data demonstrate that targeting the glycans of HCV envelope proteins is a promising approach in the development of antiviral therapies to combat a virus that is a major cause of chronic liver diseases. Furthermore, CV-N is a new invaluable tool to further dissect the early steps of HCV entry into host cells.     

In vitro evaluation of the antiviral effects of the homeopathic preparation Gripp-Heel on selected respiratory viruses

Gripp-Heel is a homeopathic preparation frequently used in the treatment of respiratory viral infections such as various types of influenza and the common cold. The antiviral activity of Gripp-Heel was studied in vitro on human pathogenic enveloped and nonenveloped RNA and DNA viruses. Before the antiviral assays, in vitro cytotoxicity of Gripp-Heel was determined with cells used for the infection experiments (HeLa, HEp-2, MDCK, BGM) as well as with mitogen-stimulated peripheral blood mononuclear leukocytes. A concentration of 0.5 of the commercially available product slightly reduced cell viability and proliferative capacity, and experiments on antiviral activity were determined starting with a dilution of 0.2 of the commercially available product. The antiviral activity was determined against a broad panel of enveloped and nonenveloped DNA and RNA viruses with plaque reduction assay, cytopathogenic assays, virus titrations, analysis of the viral proteins in virus-specific enzyme immunoassays, and haemagglutination tests. Control substances were acyclovir (10 microg/mL), ribavirin (6 microg/mL), and amantadine hydrochloride (5 microg/mL), depending on the virus type. Gripp-Heel demonstrated dose-dependent in vitro activity (significant reductions of infectivity by 20% to 40%) against Human herpesvirus 1, Human adenovirus C serotype 5, Influenza A virus, Human respiratory syncytial virus, Human parainfluenza virus 3, Human rhinovirus B serotype 14, and Human coxsackievirus serotype A9. The mechanisms of this antiviral activity are still unclear, but type I interferon induction might be a possible explanation. Further research on this homeopathic preparation seems warranted.