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.

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 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 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 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 Neutrophils Play an Essential Role in Cooperation with Antibody in both Protection against and Recovery from Pulmonary Infection with Influenza Virus in Mice

2008 ◽  
Vol 82 (6) ◽  
pp. 2772-2783 ◽  
Author(s):  
Haruo Fujisawa
Keyword(s):  
Influenza Virus ◽  
Early Phase ◽  
Influenza A ◽  
Essential Role ◽  
Late Phase ◽  
Passive Transfer ◽  
Plateau Phase ◽  
Virus Propagation ◽  
Virus Elimination

ABSTRACT The role of polymorphonuclear leukocytes (PMN) in protection in the early phase and recovery in the late phase of influenza A virus infection was investigated by the depletion of PMN in, and passive transfer of anti-influenza virus antiserum to, mice with pulmonary infections. The depletion of PMN in normal mice by treatment with monoclonal antibody RB6-8C5 both increased the mortality rate and pulmonary virus titers from the early to the late phase after infection and delayed virus elimination in the late phase. The passive transfer of the antiserum to normal mice before or after infection abolished pulmonary virus propagation in the early phase, during 3 days, or rapidly decreased high virus titers in the plateau phase, on days 3 to 5, as well as accelerated virus elimination in the late phase, on day 7, after infection, respectively. The passive transfer of the antiserum to PMN-depleted mice could neither prevent the more rapid virus propagation in the early phase, diminish the higher virus titers in the plateau phase, nor accelerate the markedly delayed virus elimination in the late phase after infection in comparison to those for controls. The antibody responses to the virus began to increase on day 7 after infection in normal and PMN-depleted mice. The prevention of virus replication, cytotoxic activity in virus-infected cell cultures, and phagocytosis of the virus in vitro by PMN were all augmented in the presence of the antiserum. These results indicate that PMN play an essential role in virus elimination in both protection against and recovery from infection, in cooperation with the antibody response.

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 EFFECT OF AMMONIUM SALTS UPON INFLUENZA VIRUS MULTIPLICATION IN VITRO

VIRUS ◽  
1957 ◽  
Vol 7 (6) ◽  
pp. 381-387 ◽  
Author(s):  
NAOKI HIGO ◽  
TAMOTSU KANEKO ◽  
NOBUO KOBAYASHI ◽  
SHYOZO NAKAJIMA ◽  
YUICHIRO SASAKI
Keyword(s):  
Influenza Virus ◽  
Virus Multiplication ◽  
Ammonium Salts

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 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 GROWTH CHARACTERISTICS OF INFLUENZA VIRUS

1954 ◽  
Vol 100 (4) ◽  
pp. 329-339 ◽  
Author(s):  
W. Wilbur Ackermann ◽  
Hunein F. Maassab
Keyword(s):  
Influenza Virus ◽  
Host Cell ◽  
Sulfonic Acid ◽  
Chorioallantoic Membrane ◽  
Biochemical Process ◽  
Cholera Vibrio ◽  
Infectious Form ◽  
Viral Development ◽  
Viral Enzyme

An analysis of the growth curve obtained in vitro for influenza virus in chorioallantoic membrane has been made using two known viral inhibitors, methoxinine and α-amino-α p-methoxyphenylmethanesulfonic acid. The action of these compounds has been shown to be specific for particular phases of viral development. Further, this action is sequential indicating that the inhibited phases do not occur concurrently. The sulfonic acid interferes with the initiation of infection and with the release of newly formed virus from the host-cell. The former effect is dependent upon the relative concentrations of viral inoculum and sulfonic acid. The latter effect is prevented by the action of the receptor-destroying enzyme obtained from cholera vibrio extracts. It is proposed that a function of the viral enzyme is to facilitate the release of virus from the host-cell. The intracellular concentration of virus necessary to initiate this release is increased in the presence of α-amino-α p-methoxyphenylmethanesulfonic acid. It was found that infection can be initiated in the presence of methoxinine under which condition viral increase is disallowed. The function in viral development of one biochemical process which is inhibited by methoxinine was found to be completed at an interval after initiation of infection and before the appearance of the infectious form of the virus. It is possible to define four stages of viral development in terms of sensitivity to methoxinine and α-amino-α-p-methoxyphenylmethanesulfonic acid.

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