scholarly journals INHIBITION OF INFLUENZA VIRUS MULTIPLICATION BY ALKYL DERIVATIVES OF BENZIMIDAZOLE

1953 ◽  
Vol 98 (3) ◽  
pp. 219-227 ◽  
Author(s):  
Igor Tamm ◽  
Karl Folkers ◽  
Frank L. Horsfall
Keyword(s):  
Influenza Virus ◽  
Latent Period ◽  
Chorioallantoic Membrane ◽  
Virus Multiplication ◽  
Influenza B Virus ◽  
Influenza B ◽  
In Ovo ◽  
Alkyl Derivatives ◽  
Rate Of Increase

At a concentration of 0.0026 M, 2,5-dimethylbenzimidazole caused a number of alterations in the first cycle of multiplication of influenza B virus, Lee strain, in chorioallantoic membrane cultures in vitro. As determined by infectivity titrations in ovo on the membrane per se, the following alterations were observed: The duration of the latent period was increased by 80 per cent. The rate of increase in titer during the incremental period was somewhat decreased. The yield of virus was decreased by about 99 per cent. When the compound was added to membrane cultures at various periods before or after inoculation with the virus, the following findings were obtained: On addition before or along with the virus, the substance caused about 99 per cent inhibition of multiplication. When added during the first 2 hours after inoculation, the compound caused inhibition of a degree which was inversely proportional to the time of addition. When added 3 to 8 hours after inoculation, the substance caused about 80 per cent inhibition. When added after the end of the latent period, no definite inhibition was obtained in the first cycle of multiplication. These results are interpreted as indicating that 2,5-dimethylbenzimidazole acts by reducing the rate of biosynthetic mechanisms necessary for the reproduction of influenza virus particles.

scholarly journals INHIBITION OF INFLUENZA VIRUS MULTIPLICATION BY ALKYL DERIVATIVES OF BENZIMIDAZOLE

1953 ◽  
Vol 98 (3) ◽  
pp. 229-243 ◽  
Author(s):  
Igor Tamm ◽  
Karl Folkers ◽  
Frank L. Horsfall
Keyword(s):  
Influenza Virus ◽  
Culture Medium ◽  
Chorioallantoic Membrane ◽  
Virus Multiplication ◽  
Influenza B Virus ◽  
Influenza B ◽  
Experimental Conditions ◽  
Virus Reproduction ◽  
B Virus

The activity of compounds which inhibit the multiplication of influenza virus can be measured in chorioallantoic membrane cultures in vitro by means of hemagglutination titrations on the medium. Studies on the reproducibility of virus reproduction in membrane cultures have revealed the major variables which affect the results and thus have led to the development of a precise technique. Under strictly controlled experimental conditions, the extent of reproduction of the virus in membrane cultures is predictable within narrow limits of variation. With 105.5 EID50 of influenza B virus, Lee strain, and 5.75 cm.2 of chorioallantoic membrane per ml., the ratio of infective virus particles to susceptible allantoic cells appears to be approximately 1:28. Under these conditions, the evidence indicates that two cycles of multiplication occur and nearly maximal hemagglutination titers are found with culture medium at 36 hours. The extent of the deviation in the absolute titer in different experiments was only 0.112 log unit. At a concentration of 0.0017 M, 2,5-dimethylbenzimidazole caused inhibition of the multiplication of influenza B virus, Lee strain, which persisted for at least 70 hours as measured by hemagglutination titrations on the culture medium. The degree of inhibition was closely comparable to that demonstrated by infectivity titrations on the membrane at the end of the first cycle of virus reproduction (1).

scholarly journals INHIBITION OF INFLUENZA VIRUS MULTIPLICATION BY ALKYL DERIVATIVES OF BENZIMIDAZOLE

1953 ◽  
Vol 98 (3) ◽  
pp. 245-259 ◽  
Author(s):  
Igor Tamm ◽  
Karl Folkers ◽  
Clifford H. Shunk ◽  
Dorothea Heyl ◽  
Frank L. Horsfall
Keyword(s):  
Influenza Virus ◽  
Inhibitory Activity ◽  
Imidazole Ring ◽  
Virus Multiplication ◽  
Influenza B Virus ◽  
Influenza B ◽  
Active Compounds ◽  
Alkyl Derivatives ◽  
Derivatives Of

The degree of inhibition of multiplication of influenza B virus, Lee strain, in membrane cultures in vitro appears to be directly related to the concentration of the inhibitory compounds used in this investigation. With each of the alkyl derivatives of benzimidazole, evidence for such a relationship was obtained in the range between 60 and 90 per cent inhibition of virus multiplication. Alteration of the structure of benzimidazole by substitution of alkyl radicals at various positions in either the benzene or the imidazole ring resulted in diverse differences in the capacity to inhibit influenza virus multiplication in vitro. Minor increases in inhibitory activity resulted when one to three methyl groups were introduced at certain positions in the molecule. Marked increases in inhibitory activity were achieved by more extensive substitution in either the benzene or the imidazole ring. The position and nature of substituent groups appeared to be of decisive importance. Among the more highly active compounds were 2,4,5,6,7-pentamethyl-benzimidazole, 5,6-diethylbenzimidazole, and 2-ethyl-5-methylbenzimidazole. Further extension of the alkyl chain at position 2 caused no significant change in the inhibitory activity of the derivative. The most active compounds studied caused 75 per cent inhibition of Lee virus multiplication in membrane cultures in vitro at concentrations of approximately 0.0002 M. Some of the implications of these findings are discussed.

scholarly journals RELATIONSHIP BETWEEN STRUCTURE OF BENZIMIDAZOLE DERIVATIVES AND SELECTIVE VIRUS INHIBITORY ACTIVITY

1961 ◽  
Vol 113 (4) ◽  
pp. 625-656 ◽  
Author(s):  
Igor Tamm ◽  
Rostom Bablanian ◽  
Marjorie M. Nemes ◽  
Clifford H. Shunk ◽  
Franklin M. Robinson ◽  
...  
Keyword(s):  
Inhibitory Activity ◽  
Cell Damage ◽  
Chorioallantoic Membrane ◽  
Virus Multiplication ◽  
Influenza B Virus ◽  
Influenza B ◽  
Cytopathic Effects ◽  
B Virus

The virus inhibitory activity and selectivity of certain benzimidazole, benzotriazole, and naphthimidazole derivatives were determined with influenza B and polio type 2 viruses. Among the sixty-five compounds examined, several were highly active inhibitors of influenza B virus multiplication in the chorioallantoic membrane in vitro. The following compounds, listed in order of increasing inhibitory activity, were more than 100 times as active as benzimidazole: 5-(4'-toluenesulfonamido)-benzimidazole, 5-hydroxybenzotriazole-4-carboxy-α-naphthylamide, 4,5,6-trichlorobenzotriazole, 5-(3',4'-dichlorobenzenesulfonamido)-benzimidazole, 5-(3',4'-dichlorobenzenesulfonamido) - 1 - (3'',4'' - dichlorobenzenesulfonyl)-benzimidazole, 4-(p-chlorophenylazo)-5-hydroxybenzotriazole, and 4,5,6,7-tetrachlorobenzotriazole. However, none showed high selectivity. Of the sixty-five compounds studied with influenza virus, twenty-five were also examined with poliovirus type 2 in monkey kidney cells in vitro. Included in this group were five of the seven most active inhibitors of influenza virus, listed above. All five were more than 100 times as active in inhibiting poliovirus multiplication as the reference compound. In addition to these, two other compounds were highly active: 2-(α-hydroxybenzyl)-benzimidazole (HBB), and 2-(α-hydroxybenzyl)-5-chlorobenzimidazole, with relative inhibitory activities of 78 and 130, respectively. These two compounds, and the much less active 5,6-dichloro derivative of HBB, were the only ones which showed no, or only slight, toxic effects on cells at concentrations sufficient to cause considerable inhibition of poliovirus multiplication. Furthermore, HBB and the 5-chloro derivative were the only compounds which caused significant inhibition of the cytopathic effects of poliovirus. HBB, and its 5-chloro and 5,6-dichloro derivatives had no effect on the multiplication of influenza B virus in the chorioallantoic membrane. In addition, HBB failed to inhibit influenza B virus multiplication and cytopathic effects in monkey kidney cells. Inhibition of poliovirus-induced cell damage by HBB was characterized by the following features: the curves relating reduction in virus yield or cytopathic effects to concentration of the compound followed an approximately parallel course; somewhat higher concentrations were required to inhibit virus-induced cell damage than to reduce virus yield. HBB suppressed viral cytopathic effects for a period of time which varied directly with the concentration of compound, and inversely with the size of virus inoculum. The development of virus-induced cell damage in treated cultures on prolonged incubation was not due to inactivation of HBB. The inhibitory effect of HBB on virus-induced cell damage was reversible by removal of the compound. HBB inhibited viral cytopathic effects when given during the exponential increase phase in virus multiplication. Inhibition of virus-induced cell damage by HBB was demonstrated by photomicrographs. HBB did not inactivate the infectivity of poliovirus type 2.

scholarly journals INFLUENZA VIRUS MULTIPLICATION IN THE CHORIOALLANTOIC MEMBRANE IN VITRO: KINETIC ASPECTS OF INHIBITION BY 5, 6-DICHLORO-1-ß-D-RIBOFURANOSYLBENZIMIDAZOLE

1954 ◽  
Vol 100 (6) ◽  
pp. 541-562 ◽  
Author(s):  
Igor Tamm ◽  
David A. J. Tyrrell
Keyword(s):  
Influenza Virus ◽  
Chorioallantoic Membrane ◽  
Inhibitory Effect ◽  
Virus Multiplication ◽  
Benzimidazole Derivatives ◽  
Similar Degree ◽  
Virus Concentration ◽  
Virus Particles ◽  
Rate Of Increase

A procedure is described for kinetic studies on the multiplication of Lee virus in the chorioallantoic membrane in vitro employing the hemagglutination technique for measurement of virus concentration. A linear relationship was found between the logarithm of virus adsorbed and the amount of membrane used. Of the virus adsorbed less than 10 per cent could be recovered from the membrane. Of the recoverable virus 90 per cent was eliminated by specific immune serum. Lee virus was adsorbed by the allantoic and chorionic layers of the membrane to a similar degree. Multiplication occurred in both layers and to a similar extent. When 107.66 EID50 of Lee virus was inoculated per 2.9 cm.2 of chorioallantoic membrane, the ratio of infectivity to hemagglutination titer in the yield was low, although the rate of appearance of virus particles was not diminished despite the large inocula. Virus produced in membranes was liberated rapidly and continually into the medium. 5,6-Dichloro-1-ß-D-ribofuranosylbenzimidazole (DRB), 0.000055 M, prolonged the latent period by more than 100 per cent. The rate of increase during the period of rapid rise was similar in the presence or absence of DRB, but the yield was markedly reduced at the end of this period in the presence of DRB. The amount of the virus in the membranes continued to rise in the presence of DRB and eventually approached the maximal levels reached much earlier in the controls. Measurement of the amount of virus in the media indicated a greater degree of inhibition than did measurement in the membranes. Comparative studies with two benzimidazole derivatives on the dependence of the inhibitory effect on the time of addition of the compound showed that processes which could be inhibited by DRB were of shorter duration than those inhibited by 2,5-dimethylbenzimidazole (MB). With MB the relationship between the time of addition and the inhibitory effect was similar both for virus and for soluble complement-fixing antigen; with DRB the inhibitable processes were of shorter duration for the complement-fixing antigen than for virus particles. DRB was not only 35 times more active on a molar basis but also was more selective in its action than MB. DRB interfered with processes which preceded the emergence of either soluble complement-fixing antigen or virus particles. Some of the implications of these findings are discussed in relation to the mechanism of inhibition of influenza virus multiplication by benzimidazole derivatives.

scholarly journals INHIBITION OF INFLUENZA VIRUS MULTIPLICATION BY N-GLYCOSIDES OF BENZIMIDAZOLES

1954 ◽  
Vol 99 (3) ◽  
pp. 227-250 ◽  
Author(s):  
Igor Tamm ◽  
Karl Folkers ◽  
Clifford H. Shunk ◽  
Frank L. Horsfall
Keyword(s):  
Influenza Virus ◽  
Influenza A ◽  
Benzimidazole Derivative ◽  
Chorioallantoic Membrane ◽  
Inhibitory Effect ◽  
Virus Multiplication ◽  
Cellular Growth ◽  
Tissue Proliferation ◽  
Fresh Culture Medium

Chloro derivatives of benzimidazole were found to be 2 to 3 times more active than corresponding methyl derivatives in causing inhibition of Lee virus multiplication in chorioallantoic membrane cultures in vitro. The most active benzimidazole derivative thus far tested is 5,6-dichloro-1-ß-D-ribofuranosylbenzimidazole (DRB); it caused 75 per cent inhibition of Lee virus multiplication in membrane cultures at a concentration of 0.38 x 10–4 M. On the other hand, 5,6-dimethyl-1-alpha;-D-ribofuranosylbenzimidazole, the moiety present in vitamin B12, failed to inhibit Lee virus multiplication at a concentration of 35 x 10–4 M. Other N-glycosides of 5,6-dichlorobenzimidazole were considerably less active than DRB. In single cycle experiments, the degree of inhibition of Lee virus multiplication by DRB in membrane cultures was not dependent on the amount of virus in the inoculum. This compound did not inactivate the infectivity of extracellular Lee virus, had no effect on virus-erythrocyte interaction, did not interfere with the adsorption of the virus by the host tissue, nor affect the release of newly formed virus from the membrane. The inhibitory effect of DRB on Lee virus multiplication, in contrast to that of 2,5-dimethylbenzimidazole, persisted after transfer of infected membranes into fresh culture medium not containing the compound. Both DRB and the 2,5-dimethyl compound caused 99 per cent inhibition of Lee virus multiplication without affecting oxygen uptake of the membrane. Tissue proliferation of membrane pieces in roller tube culture was not significantly affected by DRB at inhibitory concentration, whereas at equivalent concentration the 2,5-dimethyl compound did restrict cellular growth. At higher concentrations, both compounds caused retardation of cell proliferation. This effect was reversible on removal of either compound from the medium. The multiplication of several strains of influenza A and B viruses, i.e. Lee, MB, PR8, and FM1, was inhibited to the same degree by each of the two compounds; DRB was 35 times more active than the 2,5-dimethyl compound relative to each of the strains. DRB caused inhibition of Lee virus multiplication in intact embryonated chicken eggs and in mice without causing significant signs of toxicity in either host. Some of the implications of these findings are discussed in relation to the mechanism of the inhibition of influenza virus multiplication.

scholarly journals GROWTH CHARACTERISTICS OF INFLUENZA VIRUS

1955 ◽  
Vol 102 (4) ◽  
pp. 393-402 ◽  
Author(s):  
W. Wilbur Ackermann ◽  
Hunein F. Maassab
Keyword(s):  
Influenza Virus ◽  
Latent Period ◽  
Growth Curve ◽  
Chorioallantoic Membrane ◽  
Growth Characteristics ◽  
Stages Of Development ◽  
Viral Inhibitor ◽  
Site Of Action ◽  
Two Stages

A further analysis of the growth curve obtained in vitro for influenza virus in chorioallantoic membrane has been made using the viral inhibitor p-fluorophenylalanine. It has been found that p-fluorophenylalanine is phase-specific, does not interfere with the initiation of infection but rather acts during the productive period of the infectious sequence. The site of action of this fluoroderivative has been described relative to the site of action of methoxinine. By the use of this inhibitor in combination with methoxinine, it has been possible to recognize two stages of development which were not discernible from the usual growth curve. In a multicellular culture these two reactions, A and B, occur for the most part simultaneously, during the latent and productive periods. Reaction A is inhibited by methoxinine, but not by fluorophenylalanine. It begins early in the latent period and can proceed independent of reaction B. Reaction B is inhibited by fluorophenylalanine, but not by methoxinine, and it cannot proceed unless reaction A is proceeding or has operated for some prior interval.

scholarly journals ON THE ROLE OF RIBONUCLEIC ACID IN ANIMAL VIRUS SYNTHESIS

1960 ◽  
Vol 111 (3) ◽  
pp. 339-349 ◽  
Author(s):  
Igor Tamm ◽  
Marjorie M. Nemes ◽  
Suydam Osterhout
Keyword(s):  
Influenza Virus ◽  
Chorioallantoic Membrane ◽  
Inhibitory Effect ◽  
Virus Multiplication ◽  
Point Of View ◽  
Host Cells ◽  
Animal Virus ◽  
Host Cell Proteins

Adenosine, but not guanosine, was capable of blocking the inhibitory effect of 5,6-dichloro-1-ß-D-ribofuranosylbenzimidazole (DRB) on influenza virus multiplication in the chorioallantoic membrane in vitro. At virus inhibitory concentrations DRB caused marked inhibition in uptake of adenosine-8-C14 into RNA of uninfected host cells, but it had little effect on uptake of C14-L-alanine into host cell proteins or on cellular oxygen consumption. The activity of DRB in inhibiting multiplication of the DNA-containing adenovirus was similar to its inhibitory activity on multiplication of the RNA-containing influenza virus. These and earlier results are discussed from the point of view of the important role of RNA in the reproduction of DNA-containing viruses.

scholarly journals Inhibitory effects of aprotinin on influenza A and B viruses in vitro and in vivo

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Eun-Jung Song ◽  
Erica Españo ◽  
Sang-Mu Shim ◽  
Jeong-Hyun Nam ◽  
Jiyeon Kim ◽  
...  
Keyword(s):  
Influenza Virus ◽  
Influenza A ◽  
Lethal Dose ◽  
Influenza Viruses ◽  
Influenza B Virus ◽  
Influenza B ◽  
Inhibitory Effects ◽  
Compound Libraries ◽  
Wide Range

AbstractInfluenza viruses cause significant morbidity and mortality worldwide. Long-term or frequent use of approved anti-influenza agents has resulted in drug-resistant strains, thereby necessitating the discovery of new drugs. In this study, we found aprotinin, a serine protease inhibitor, as an anti-influenza candidate through screening of compound libraries. Aprotinin has been previously reported to show inhibitory effects on a few influenza A virus (IAV) subtypes (e.g., seasonal H1N1 and H3N2). However, because there were no reports of its inhibitory effects on the other types of influenza viruses, we investigated the inhibitory effects of aprotinin in vitro on a wide range of influenza viruses, including avian and oseltamivir-resistant influenza virus strains. Our cell-based assay showed that aprotinin had inhibitory effects on seasonal human IAVs (H1N1 and H3N2 subtypes), avian IAVs (H5N2, H6N5, and H9N2 subtypes), an oseltamivir-resistant IAV, and a currently circulating influenza B virus. We have also confirmed its activity in mice infected with a lethal dose of influenza virus, showing a significant increase in survival rate. Our findings suggest that aprotinin has the capacity to inhibit a wide range of influenza virus subtypes and should be considered for development as a therapeutic agent against influenza.

scholarly journals The memberane Piece Technique for in Vitro Infectivity Titrations of Influenza Virus

1957 ◽  
Vol 55 (3) ◽  
pp. 434-456 ◽  
Author(s):  
N. B. Finter ◽  
P. Armitage
Keyword(s):  
Influenza Virus ◽  
Influenza A ◽  
Technical Assistance ◽  
Allantoic Fluid ◽  
Test Tube ◽  
Influenza B Virus ◽  
Influenza B ◽  
Experimental Conditions ◽  
Set Up

1. The membrane piece technique for in vitro titrations of the infectivity of influenza virus is described. Rectangles of shell, about 8 × 25 mm., with the chorio-allantoic membrane still attached (membrane pieces) are cut from thirteenth-day fertile eggs. One piece in a test-tube with glucose-buffered salt solution forms an individual assay unit. Five or more tubes are inoculated with each virus dilution. After incubation at 37° C. for 72 hr., with agitation for the first 24 hr. the fluid in each tube is tested for haemagglutinins. From the results at each dilution, an estimate of the 50% membrane piece (MP50) infectivity titre is obtained.2. Six hundred assay units, with pieces cut from twenty eggs, can be set up by two workers in 1 hr. and used for titration of between three and twenty-four individual virus preparations, depending on the reliability desired for the 50% end-point estimates.3. With the D.S.P. and PR 8 strains of influenza A virus, the MP50 titres parallel the EID50 titres from egg titrations, but are eight times and twenty times lower, respectively. The MP50: EID50 ratio is the same for various preparations of the same strain, including standard allantoic fluid and chorio-allantoic membrane virus, incomplete virus, and inactivated (heated) allantoic fluid virus. Preliminary experiments with Lee influenza B virus show that slightly different experimental conditions are required, and the MP50 titres are about fifty times less than the EID50 titres.4. Consistent results have been obtained on titration of samples of the same virus preparation on a number of occasions over a period of several months.5. A large number of membrane pieces can be used to test each virus dilution, and sampling variations in the MP50 estimates thus made quite small. Statistical data on the reliability of a 50 % titration result, and on the minimum significant differences between two end-points, are given for different values of n, the number of membrane pieces used to test each virus dilution, and of d, the log dilution step.We are grateful to Mr J. Collins for invaluable technical assistance, and also to Miss I. Allen for help with the computations.

scholarly journals GROWTH CHARACTERISTICS OF INFLUENZA VIRUS: THE INFLUENCE OF A SULFONIC ACID

1954 ◽  
Vol 99 (2) ◽  
pp. 105-117 ◽  
Author(s):  
W. Wilbur Ackermann ◽  
Hunein F. Maassab
Keyword(s):  
Influenza Virus ◽  
Latent Period ◽  
Early Phase ◽  
Sulfonic Acid ◽  
Cellular Membrane ◽  
Virus Multiplication ◽  
Growth Characteristics ◽  
Common Character

The growth characteristics of influenza virus in an isolated tissue maintained in vitro have been described. When compared with previously reported results using the embryonate egg, a considerably shorter latent period was observed. The release or liberation of the virus occurred throughout a period of many hours. There was no evidence of a general "burst" phenomenon, and the destruction of a cellular membrane did not seem to be essential to or concomitant with the release of virus. An early phase in the development of virus was described which is sensitive to the action of α-amino-p-methoxyphenylmethanesulfonic acid and it is by virtue of this that virus multiplication is prevented. If this phase was allowed to go on to completion, replication of virus occurred even in the presence of the sulfonic acid, but the release of virus from the tissue was impaired. It is suggested that the sulfonic acid may interfere with the adsorption or penetration of the virus and that the initiation of infection and the liberation of new virus may be processes which share some common character.

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