Several kinds of ammonium salt inhibited the propagation of influenza virus in vitro in a very low concentration (10-3M or less). Out of several kinds of ammonium salt, NH4Cl was selected and its effect upon influenza virus multiplication in vitro was observed. Ammonium chloride did neither prevent the adsorption of the virus to the host cells nor inactivate the virus directly. The earlier the addition of ammonium chloride after virus infection, the stronger inhibitory effect was observed. Even when ammonium chlorid was removed from the cultivated tissue by washing, the virus propagation was very small even after 24 hour of virus infection, but the multiplication occurred in later stages and after 48 hour virus titer was almost the same as the control without ammonium chloride. Furthermore. on the previously with ammonium chloride treated host tissue virus multiplication was much smaller 24 hour after infection than that of control, but in later stage (48th hour after infection) virus propagated on the both treated-and nontreated host cell to the almost same titer. Therefore, ammonium chloride may inhibit probably the virus multiplication by damaging normal host cells on which the virus would be propagated. Such a damage of the normal cells was reversible, although its convalescence was delayed for a long time.
Many antivirals which will inhibit the growth of influenza virus in tissue culture system have been isolated in this laboratory from various streptomyces filtrates. Though almost all of them, except myxoviromycin, did not have any chemotherapeutic effect against experimental influenza in mice, site of action of these compounds have been studied in order to get further information on the diverse action of various antivirals. The result obtained in such studies are the subject of this series paper. In this particular communication, the site of action of streptothricin-3 was studied. The host-virus system was maintained in an Erlenmeyer flask using 3ml of modified Hanks' solution as medium. Each flask contained 400mg of chorioallantoic membrane from 15 days embryonated egg. The PR8 strain was used thoughout the work. The amount of virus in the fluid was estimated both by determining the infectious titer in tissue culture system and heniagglutinin. The result will be summarized as follows: 1) One mg/ml of streptothricin-3 was the minimum concentration which inhibited the viral growth completely when 105 TCID50 of virus was used as the inoculum. During the incubation at 37°C, the degradation of the substance was almost 30% within first 6 hrs. 2) Depending upon the inoculum size, the effect of the substance on the growth of virus varied. 3) Neither virocidal activity nor inhibition of the adsorption stage to the host has been observed with this substance. 4) The course of viral development was influenced even when the antibiotic was added as late as 10 hrs. after infection. 5) Incomplete reversibility of the action of the substance to the host cell was found when normal chrioallantoic membrane was treated with the substnce as long as 7 hrs. before infection.
In the previous communication, the mode of action of streptothricin-3 on the growth of influenza virus has been studied in a tissue culture system using chorioallantoic membrane as host cells. In the studies to be reported here, chartreusin, chemically quite different antivirals from streptothricin-3 has been tested just in the same manner as was done in a previous examination. The isolation of crystalline chartreusin from a filtrate of a streptomyces designated as G72, and the mode of action of the agent on the growth of T3 phage has been already reported by Kaneko. The result of this study will be summarized as follows: 1) The potency of the substance did not destroyed when incubated at 37°C for 24 hrs., in the tissue culture system employed. 2) As low as 25mcg/ml was the minimum inhibitory concentration when 105TCID50 of the PR8 virus was used as inoculum. However, with concentration of 12.5mcg/ml, comparable good growth to the control was obtained. In other words, changing small range of concentrations gave considerable difference on the inhibition rate. This fact was also observed by Anzai, independently. 3) Sensitivity phase during the course of virus development to this agent was examined and the addition of the substance 3 hrs. after infection did not inhibit the virus growth, while the addition at 0 and 1 hr. gave complete inhibition. This finding will suggest that chartreusin sensitive stage may exist earlier than that of streptothricin-3. 4) Depending upon the inoculum size, the effect of the substance on the growth of virus varied. This fact, however, has to be studied more throughly in a light of Anzais work, in which he found the contrary result by different analytical procedures. 5) Reversibility of the action of the substance to the host cell was observed when normal chorioallantoic membrance was treated with the substace for 5 hrs. before infection. All of the evidence illusrated here may suggest the different site of action of the afore-mentioned 2 substances, i.e. streptothricin-3 and chartreusin and will be discussed further in detail in an accompanying paper.
From the results of two preceding papers, it seemed advisable to investigate the effect of two antivirals i.e. streptothricin-3 and chartreusin, on the growth of PR8 virus, comparatively at the same time. Canavanine sulfate in addition was used as an inhibitor in order to analyse the reacting phase of the afore-mentioned antivirals, because the stage where canavanine acts coincides with that of p-fluorophenyl-alanine and may presumably has the effect on the protein synthesis of the virus. First experiment was performed with these three antivirals in order to determine the effect of time of addition on virus growth. Second experiment was conducted by examining the differential response of the viral growth every 2 hours. By means of this response, the late effect of each antivirals on the virus growth has been measured accurately and in simple fashion. The experiment was as follows; i.e. 6 hrs. after infection, similarly infected membranes were put in the new flasks which contained these antivirals respectively, and each membrane was transferred to the other new antiviral containing medium every two hours. The production of hemagglutinin was tested in each flask. All of the result obtained in the above experiment seems to suggest that the sensitive stage to these 3 antivirals are different in each other and in the order of chartreusin, streptothricin-3 and Canavanine sulfate, from earlier to later.
We studied if egg-adapted lines of influenza virus FM1 are non-pathogenic to mice but grow in the lung of mice. We saw that egg-adapted lines FM1 which are subculturer in eggs for many generations survived in the lung of mice for some days after intranasal inoculation, but we did not recognize the production of egg-adapted lines of influenza virus FM1 in the lung of mice.