Virus-inhibiting properties of the carbonyl-conjugated pentaene roseofungin

The antiinfluenza activity of roseofungin, a polyenic macrolide antibiotic was studied in vitro on surviving fragments of the chick embryo chorionallantoic membranes and in ovo on growing chick embryos. It was shown that the antibiotic activity against influenza A and B viruses was sufficiently high. The activity of roseofungin against influenza A virus did not differ from that of remantadin, the most active inhibitor of influenza virus reproduction. However, the activity of roseofungin against influenza B virus was an advantage of this antibiotic over remantadin, which had practically no effect on this virus type. A statistically significant protective effect of roseofungin (p less than 0.05) was shown on the animals with experimental influenza. The study on the antiviral activity of roseofungin against the DNA-containing variolovaccine virus revealed that it markedly inhibited the plague reduction. Roseofungin had a pronounced inhibitory effect on cell neoplastic transformation induced by the RNA-containing oncogenic virus of Rous sarcoma.     

Electron absorption and fluorescence spectra of carbonyl--conjugated pentaenes in various media]

It was found that fluorescence intensity of carbonyl-conjugated pentaens of flavofungin flavopentin and brunefungin in solutions depended on the solvent polarity and specific interactions of the antibiotics with the solvents. Addition of cholesterol into the aqueous solutions of these antibiotics resulted in a hypsochrome shift in their absorption spectra and increased fluorescence intensity. The antibiotics were found by their fluorescence in the yeast cells sensitive to them when their content was close to the concentrations resulting in the cell lysis. Low concentrations of the antibiotics resulted in changed localization of the yeast self fluorescence.     

蛋氨酸脑啡肽（MEK）抗B型流感病毒感染作用的研究

研究MEK抗B型流感病毒感染的作用。采用MDCK细胞和9～10日龄鸡胚,按不同的顺序加入不同剂量MEK和B型流感病毒,共培养72 h后做血凝实验。所有加入B型流感病毒的MDCK细胞均培养出病毒,HA滴度为1：64。在鸡胚尿囊腔中,先注入MEK孵育24 h后,再注入B型流感病毒的鸡胚也培养出病毒,HA滴度为1：6.8,与病毒对照组比较P〈0.01,有统计学意义。实验结果未见MEK直接抗B型流感病毒感染MDCK细胞株的作用,但可见MEK抗B型流感病毒感染鸡胚的作用。     

Virucidal effect of sodium p-chloromercuribenzoate on influenza viruses attributable to inhibition of virus particle-associated RNA-dependent RNA polymerase.

Sodium p-chloromercuribenzoate (PCMB) caused a noticeable reduction of infectivity of prototype strains of type A and Lee strain of type B influenza viruses at concentrations of 100 and 200 mug/ml, respectively, after an incubation at 37 C for 60 min. The virucidal effect on A/AA/2/60 (H2N2) strain was dependent on the concentration of the drug and temperature as well as on the time of incubation. The reagent exerted this effect at a concentration which induced little change in the hemagglutinating and neuraminidase activities of the virus. PCMB inhibited by 50% the virus particle-associated RNA polymerase activity of all prototype strains of type A influenza virus at about 2 mug/ml and that of Lee strain of type B influenza virus at 8.5 mug/ml. Other sulfhydryl reagent such as phenylmercuric nitrate also exhibited virucidal effect on A/AA/2/60 virus which paralleled their inhibition of the virus particle-associated RNA polymerase activity. From these results it was considered likely that the virucidal action of PCMB on influenza viruses was attributable to inhibition of the virus particle-associated RNA polymerase activity.     

Properties of the erythrocyte receptors for influenza C virus.

Properties of the receptor for influenza C virus were studied. Although the receptor for influenza C virus on chicken erythrocytes was destroyed by the homologous virion, neuraminidase activity could not be detected in any of the influenza C virus strains tested. The receptor activity of chicken erythrocytes for influenza C virus was diminished by formaldehyde treatment but not by periodate oxidation. There was a considerable variation in the pattern and the titer of hemagglutination of influenza C virus when human erythrocytes of different blood types were used; the virus agglutinated most type B erythrocytes but not type A erythrocytes. By using human type B erythrocytes, differences among strains of influenza C virus in the hemagglutinating activity were also demonstrated. These results showed that both the receptor for and the receptor-destroying activity of influenza C virus were completely different from those of influenza A or B virus and also that carbohydrates were not involved in the receptor for influenza C virus.     

Broad-spectrum antiviral effect of Agrimonia pilosa extract on influenza viruses

Influenza virus continues to emerge and re-emerge, posing new threats for humans. Here we tested various Korean medicinal plant extracts for potential antiviral activity against influenza viruses. Among them, an extract of Agrimonia pilosa was shown to be highly effective against all three subtypes of human influenza viruses including H1N1 and H3N2 influenza A subtypes and influenza B virus. The EC₅₀ value against influenza A virus, as tested by the plaque reduction assay on MDCK cells, was 14–23 μg/ml. The extract also exhibited a virucidal effect at a concentration of 160–570 ng/ml against influenza A and B viruses when the viruses were treated with the extract prior to plaque assay. In addition, when tested in embryonated chicken eggs the extract exhibited a strong inhibitory effect in ovo on the H9N2 avian influenza virus at a concentration of 280 ng/ml. Quantitative RT-PCR analysis data showed that the extract, to some degree, suppressed viral RNA synthesis in MDCK cells. HI and inhibition of neuraminidase were observed only at high concentrations of the extract. And yet, the extract's antiviral activity required direct contact between it and the virus, suggesting that its antiviral action is mediated by the viral membrane, but does not involve the two major surface antigens, HA and NA, of the virus. The broad-spectrum antiviral activity of Agrimonia pilosa extract on various subtypes of influenza viruses merits further investigation as it may provide a means of managing avian influenza infections in poultry farms and potential avian-human transmission.     

Virucidal Effect of Sodium p-Chloromercuribenzoate on Influenza Viruses Attributable to Inhibition of Virus Particle-Associated RNA-Dependent RNA Polymerase

Sodium p-chloromercuribenzoate (PCMB) caused a noticeable reduction of infectivity of prototype strains of type A and Lee strain of type B influenza viruses at concentrations of 100 and 200 μg/ml, respectively, after an incubation at 37 C for 60 min. The virucidal effect on A/AA/2/60 (H₂N₂) strain was dependent on the concentration of the drug and temperature as well as on the time of incubation. The reagent exerted this effect at a concentration which induced little change in the hemagglutinating and neuraminidase activities of the virus. PCMB inhibited by 50% the virus particle-associated RNA polymerase activity of all prototype strains of type A influenza virus at about 2 μg/ml and that of Lee strain of type B influenza virus at 8.5 μg/ml. Other sulfhydryl reagent such as phenylmercuric nitrate also exhibited virucidal effect on A/AA/2/60 virus which paralleled their inhibition of the virus particle-associated RNA polymerase activity. From these results it was considered likely that the virucidal action of PCMB on influenza viruses was attributable to inhibition of the virus particle-associated RNA polymerase activity.     

Effects of Clinacanthus siamensis leaf extract on influenza virus infection

Ethanolic extracts of 20 medicinal plants were screened for influenza virus NA inhibition and in vitro antiviral activities using MDCK cells in an MTT assay. The vaccine proteins of influenza virus A/New Caledonia/20/99 (H1N1), mouse-adapted influenza virus A/Guizhou/54/89 (A/G)(H3N2) and mouse-adapted influenza virus B/Ibaraki/2/85 (B/I) were used in the NA inhibition assay, and mouse-adapted influenza viruses A/PR/8/34 (H1N1), A/G and B/I were used in the in vitro antiviral assay. The results of the in vitro antiviral assay indicated that the A/G virus was the most susceptible and an extract of the leaf of CS possessed the highest in vitro anti-A/G virus activity (41.98%). Therefore, the A/G virus and the CS extract were selected for studying in vivo anti-influenza virus activity. BALB/c mice were treated with CS extract (100 mg/kg per day, 5 times) orally from 4 hr before to 4 days after infection. CS extract elicited significant production of anti-influenza virus IgG₁ antibody in BAW and increased mouse weight compared to oseltamivir (0.1 mg/kg per day) on day 19 or water on days 17–19 of infection. Moreover, CS extract produced a higher anti-influenza virus IgA antibody level in BAW compared to oseltamivir, and a tendency towards an increase in anti-influenza virus IgA compared to water was shown. The results suggest that CS extract has a protective effect against influenza virus infection.     

Substituted Sialic Acid Prosthetic Groups as Determinants of Viral Hemagglutination

Inhibitors of hemagglutination by type A2 influenza virus and a recently isolated strain of type B influenza virus were separated by sucrose density gradient centrifugation and agarose gel filtration from horse serum. Using selected reagents, it was demonstrated that the active substituent on the horse serum inhibitor of A2 influenza virus was 4-O-acetyl-N-acetylneuraminic acid; however, the active substituent on the inhibitor of the influenza B virus was shown to be N-acetylneuraminic acid (NANA). Sodium metaperiodate treatment of a component of horse serum resulted in a 10 to 15-fold enhancement of inhibitory activity against the type B virus, whereas the A2 inhibitor was completely destroyed. Since this enhancement did not occur with influenza B viruses isolated prior to 1965, it was considered that this sensitivity to an oxidized NANA glycoside may have been a reflection of an antigenic change which occurred at that time. The use of different virus strains and selected chemical reagents to define the important sialic acid prosthetic groups active in inhibition was described.     

Fusion and infection of influenza and Sendai viruses as modulated by dextran sulfate: a comparative study

We have directly compared the effect of two types of dextran sulfate with distinct molecular weights (500 kDa and 5 kDa) on the fusion activity and infectivity of both Sendai and influenza viruses, two lipid-enveloped viruses that differ in their routes of entry into target cells. To correlate membrane merging and infectivity MDCK cells were used as targets for the viruses in both approaches. In either case pronounced inhibition of virus–cell interactions by dextran sulfate was only observed at low pH, even though Sendai virus fuses maximally at pH 7.4. Although membrane merging could not be fully abolished, the inhibitory effect was always greater when the higher molecular weight dextran sulfate was used. The presence of this residual fusion activity, that could not be reduced even with high concentrations of agent, suggests that a limited number of binding sites for dextran sulfate may exist on the viral envelopes. The compounds also inhibited fusion of bound virions, and all results could be reproduced using erythrocyte ghosts as target membranes in the fusion assay, instead of MDCK cells. In agreement with these observations only the infectivity of influenza virus (which requires a low pH-dependent step to enter target cells) was affected by dextran sulfate, again the higher molecular weight compound showing a more pronounced inhibitory effect.     

Activity of recombinant human alpha interferon against influenza virus infection in mice

The in vivo antiviral activity of recombinant human leukocyte hybrid interferon, HuIFN-alpha AD, was examined. Results showed that this material in highly purified form did not protect mice against a lethal dose of influenza virus, although administration of natural MuIFN-alpha/beta to mice infected with a lethal dose of influenza virus had a marked protective effect. The effect of alveolar macrophages treated with IFN on influenza virus replication was examined in vitro. The antiviral activity of alveolar macrophages treated with HuIFN-alpha AD was lower than that of MuIFN-alpha/beta. It is concluded that HuIFN-alpha AD is effective in direct inhibition of influenza virus, but not in indirect inhibition mediated by alveolar macrophages or in protection of mice from influenza virus infection.     

Enzymological characteristics of avian influenza A virus neuraminidase

Neuraminidases of 18 strains of avian influenza A virus were examined by both colorimetric and fluorometric assays using fetuin and 4-methylumbelliferyl-N-Ac-alpha-D-neuraminide as substrates, respectively, to compare them with those of human influenza A and B viruses. The ratios of the neuraminidase activity of avian influenza virus measured by the colorimetric assay method to that measured by the fluorometric assay were distributed in the range of 2.4-20.3. The enzyme of avian influenza virus showed calcium-ion dependence in both assay methods. These results suggest that neuraminidase of avian influenza A virus is varies greatly from one strain to another in substrate specificity as compared with those of human influenza A and B viruses, and that some strains of avian influenza A virus have a neuraminidase with unique enzymological characteristics different from that of human influenza A virus as well as that of influenza B virus.     

Different progress of MDCK cell death after infection by two different influenza virus isolates

The effect of influenza strains A (H₃N₂) and B, isolated during the seasons of 1994 and 1995 in the Czech Republic, on MDCK cells was studied. Various concentrations of virus and conditions of nutrition were used during the cell culture. The virus replication and consequently fragmentation of genomic DNA together with cytotoxicity were investigated in the absence and presence of 10 per cent calf serum. Virus replication, regardless of type A or B, caused earlier DNA fragmentation in comparison to non-infected cells in tissue culture. The results showed that the influenza B strain had a greater cytotoxic effect on MDCK cells than influenza A. A higher infection dose of influenza A virus accelerated the onset of apoptosis; conversely, a higher infection dose of influenza B virus delayed the onset of apoptosis. The absence of serum enhanced the progress of influenza-induced apoptosis in conditions in vitro. © 1997 John Wiley & Sons, Ltd.     

A型流感病毒持续感染细胞系来源的IVpi-189病毒生物学性状的初步研究

为明确E61-24-P15 A型重组流感病毒的第189代传代子病毒(IVpi-189)是否具备流感病毒温度敏感减毒活疫苗候选株的特点,将IVpi-189病毒感染MDCK细胞,并于不同培养温度条件下培养,观察其致细胞病变效应,病毒合成、释放情况,以及不同温度条件下病毒存活时间。结果显示32℃培养温度下,IVpi-189病毒具有等同于亲代野生病毒株的诱导细胞病变能力,而当培养温度上调至38℃,IVpi-189病毒致细胞病变效果出现缓慢且程度明显减轻。空斑形成单位实验发现IVpi-189病毒在38℃培养条件下增殖能力明显下降,其原因与病毒灭活速度及子病毒释放无关,但与感染细胞病毒合成能力下降有关。上述实验结果初步证实流感病毒持续感染细胞系来源的IVpi-189病毒具有温度敏感减毒活疫苗的生物学特性,在许可培养温度条件下具有良好的增殖能力,而在非许可培养温度下,病毒增殖活性受到明显抑制。本研究为流感病毒减毒活疫苗的开发研制提供实验佐证。     

Hepatic carbamyl phosphate synthetase and ornithine transcarbamylase in mouse influenze A and influenze B infection.

In mice infected with mouse-adapted influenza A/PR/8/34, hepatic carbamyl phosphate synthetase (CPS) activity was reduced to 88%, and ornithine transcarbamylase (OTC) was reduced to 83% of control values. In mice infected with mouse-adapted B/Lee/40, CPS activity was 98% was 94% of control values. These limited reductions in enzyme activity were attributed to a nonspecific debilitating effect of acute influenzal pneumonia. These findings suggest that the pronounced reduction of CPS and OTC activities reported in Reye's syndrome in man are not a general manifestation of the severity of influenza infection.     

Antimycotic-antibiotic amphotericin B promotes influenza virus replication in cell culture

In general, antibiotics are not rated as substances that inhibit or support influenza virus replication. We describe here the enhancing effect of the polyene antibiotic amphotericin B (AmB) on influenza virus growth in Vero cells. We show that isolation rates of influenza A and B viruses from clinical samples can be dramatically enhanced by adding AmB to the culture medium. We demonstrate that AmB promotes the viral uptake and endocytic processing of the virus particles. This effect is specific for Vero and human nasal epithelial cells and was not observed in Madin-Darby canine kidney cells. The effect of AmB was subtype specific and more prominent for human seasonal influenza strains but absent for H5N1 human viruses. The AmB-enhancing effect seemed to be solely due to the viral hemagglutinin function. Our results indicate that the use of AmB may facilitate influenza virus isolation and production in Vero cells.     

Identification of a PA-Binding Peptide with Inhibitory Activity against Influenza A and B Virus Replication

There is an urgent need for new drugs against influenza type A and B viruses due to incomplete protection by vaccines and the emergence of resistance to current antivirals. The influenza virus polymerase complex, consisting of the PB1, PB2 and PA subunits, represents a promising target for the development of new drugs. We have previously demonstrated the feasibility of targeting the protein-protein interaction domain between the PB1 and PA subunits of the polymerase complex of influenza A virus using a small peptide derived from the PA-binding domain of PB1. However, this influenza A virus-derived peptide did not affect influenza B virus polymerase activity. Here we report that the PA-binding domain of the polymerase subunit PB1 of influenza A and B viruses is highly conserved and that mutual amino acid exchange shows that they cannot be functionally exchanged with each other. Based on phylogenetic analysis and a novel biochemical ELISA-based screening approach, we were able to identify an influenza A-derived peptide with a single influenza B-specific amino acid substitution which efficiently binds to PA of both virus types. This dual-binding peptide blocked the viral polymerase activity and growth of both virus types. Our findings provide proof of principle that protein-protein interaction inhibitors can be generated against influenza A and B viruses. Furthermore, this dual-binding peptide, combined with our novel screening method, is a promising platform to identify new antiviral lead compounds.     

Inhibition of sialidases from viral,bacterial and mammalian sources by analogues of 2-deoxy-2,3-didehydro-N-acetylneuraminic acid modified at the C-4 position

The inhibition of sialidase activity from influenza viruses A and B, parainfluenza 2 virus,Vibrio cholerae, Arthrobacter ureafaciens, Clostridium perfringens, and sheep liver by a range of 2-deoxy-2,3-didehydro-N-acetylneuraminic acid analogues modified at the C-4 position has been studied. All substitutions tested resulted in a decrease in the degree of inhibition of the bacterial and mammalian sialidases. For sialidases from influenza viruses A and B, on the other hand, most of the substitutions tested either had no significant effect on binding or, in the case of the basic amino and guanidino substituents, resulted in significantly stronger inhibition. The results for parainfluenza 2 virus sialidase were mostly intermediate, in that inhibition was neither significantly increased nor decreased by most of the modifications. We conclude that only the influenza A and B sialidase active sites possess acid groups correctly positioned to participate in charge-charge interactions in the region of C-4 of bound substrate, and that the C-4 binding pockets of the bacterial and mammalian sialidases examined are considerably smaller than is observed for either the influenza virus or parainfluenza virus sialidases.This paper is dedicated to the memory of Professor Dr E. Zbiral.     

Current status for influenza control

Influenza viruses are responsible for respiratory illness with significant morbidity and mortality. To curb the disease, two-pronged attack on the virus, therapeutic and prophylactic, is being actively pursued. The therapeutic use of existing anti-influenza drugs, such as amantadine and rimantadine, is limited by their significant adverse side effect, emergence of resistant viral strains, and lack of activity against influenza B virus. A new class of antiviral agents designed to inhibit influenza neuraminidase are currently under active development for use in the prophylaxis and treatment of influenza A and B virus infections. Two of these compounds, zanamivir (GG167) and GS4104 have reached clinical trials. Limitations in the effectiveness and application of inactivated vaccines have stimulated development of alternative approaches to influenza immunization. One such approach is a live, intranasally administered vaccine, attenuated by cold-adaptation of a master strain with subsequent genetic reassortment with circulating wild-type strains. Recently developed reverse-genetics techniques have made it possible to use RNA viruses as vector. Besides DNA viral vectors, live influenza virus vectors may emerge as a useful alternative for the vaccination against different pathogens.