scholarly journals STUDIES ON HOST-VIRUS INTERACTIONS IN THE CHICK EMBRYO-INFLUENZA VIRUS SYSTEM

1956 ◽  
Vol 103 (6) ◽  
pp. 799-822 ◽  
Author(s):  
Werner Henle ◽  
Oscar C. Liu ◽  
Kurt Paucker ◽  
Florence S. Lief
Keyword(s):  
Chick Embryo ◽  
Infectious Virus ◽  
Potassium Cyanide ◽  
Cold Room ◽  
The Status ◽  
The Media ◽  
Host Virus Interactions ◽  
Virus Interactions ◽  
Virus System ◽  
Made In

Studies have been reported concerning the relationships between virus materials found in the allantoic membranes and media of eggs deembryonated after injection of Standard (ST), heat-inactivated (37°C.) standard (ΔST), and undiluted passage (UP) seeds. It was found that the membranes always contained relatively more non-infectious hemagglutinins (NIHA) than the media and, correspondingly, the ratios between infectious virus and hemagglutinin units (ID50/HA) in the tissues were up to 1.5 log10 units lower than in the liberated progeny. These differences were seen not only following inoculation of undiluted ST, ΔST, and UP seeds, the progenies of which always contain considerable proportions of NIHA, but also when dilute ST inocula were employed which lead to the liberation of only infectious virus. Essentially similar differences in the ID50/HA ratios were observed also in the allantoic membranes and fluids obtained from growth curve experiments in the intact chick embryo employing the various types of seeds. In correlating the liberated virus materials in the media of deembryonated eggs to those in the membranes it was noted that in any given 2 hour interval during the phase of nearly constant production and release up to 10 times the quantity of infectious virus was shed as was present in the tissues at the onset of that period. In contrast, only about ¼ of the hemagglutinins were released during the same time. The viral (V) and soluble (S) complement-fixing antigens were found in the tissues but no detectable quantities were released during any 2 hour interval. The NIHA in the membranes apparently is located within the cells since it could not be released by the action of RDE. Intracellular inhibitors of hemagglutination were readily inactivated following inoculation of undiluted ST, ΔST, or UP seeds but not when ultraviolet-inactivated virus was used. The inhibitor activity decreased in proportion to the hemagglutinins produced. Transfer of infected deembryonated eggs to the cold room after production and liberation of progeny were well under way immediately halted further release but in the tissues the status quo was maintained and release was resumed on return to the 37°C. incubator. The addition of potassium cyanide to the medium of deembryonated eggs at 37°C. during the period of nearly constant production and release of virus material reduced immediately and to comparable extents the ID50 and HA titers in the tissues and liberation decreased in proportion. On removal of the cyanide 2 hours later, both titers in the tissues gradually returned to those of the untreated control eggs with a corresponding increase in liberation. The ID50/HA ratios were not affected by these manipulations. It is concluded that the NIHA in the membranes forms part of a dynamic process. An attempt has been made in the discussion to integrate the present results with previous observations concerning the formation of incomplete forms of virus and their nature and role in the infectious process.

scholarly journals STUDIES ON HOST-VIRUS INTERACTIONS IN THE CHICK EMBRYO-INFLUENZA VIRUS SYSTEM

1955 ◽  
Vol 101 (5) ◽  
pp. 461-478 ◽  
Author(s):  
Norman B. Finter ◽  
Oscar C. Liu ◽  
Werner Henle
Keyword(s):  
Influenza Virus ◽  
Chick Embryo ◽  
Host Cell ◽  
Infectious Virus ◽  
Chick Embryos ◽  
In Ovo ◽  
Virus Particles ◽  
Host Virus Interactions ◽  
Virus Interactions ◽  
Virus System

An analysis has been made of factors contributing to the von Magnus phenomenon; i.e., the emergence of increasing quantities of non-infectious hemagglutinins (NIHA) in successive passages in the allantois of chick embryos of undiluted allantoic fluids infected with influenza virus. Using the PR8 strain, the von Magnus phenomenon was pronounced when the serial seeds were obtained under conditions which permitted extensive inactivation of infectious virus during individual passages. Correspondingly, it was reduced but not abolished when precautions were taken to avoid accumulation of inactivated virus in the inocula. Thus, inactivated virus may be taken as a contributing factor. Preparations of infectious virus obtained under conditions largely excluding the presence of inactivated virus were capable of yielding some NIHA on passage as long as sufficient amounts were injected to permit each host cell to adsorb several infectious virus particles. However, the fact remains that more NIHA was found in the harvests when the inocula contained a large proportion of non-infectious virus material. Following injection of various types of seeds NIHA appeared in the allantoic fluids as soon as liberation of virus became detectable. This time relationship and the rates of release of non-infectious virus components seemed to exclude that the NIHA obtained consisted entirely of infectious virus which had been inactivated during incubation in ovo. It was apparent rather that NIHA other than that due to heat-inactivated virus was released. Correlations between the infectivities and hemagglutinating capacities of over 50 standard and undiluted passage seeds and the compositions of the harvests derived therefrom on passage without dilution indicated that the corresponding activities in the yields did not depend entirely upon the relative concentrations of infectious virus and non-infectious hemagglutinins in the inocula but that apparently different forms of NIHA were obtained in successive undiluted passages.

scholarly journals STUDIES ON HOST-VIRUS INTERACTIONS IN THE CHICK EMBRYO-INFLUENZA VIRUS SYSTEM

1955 ◽  
Vol 101 (5) ◽  
pp. 493-506 ◽  
Author(s):  
Kurt Paucker ◽  
Werner Henle
Keyword(s):  
Influenza Virus ◽  
Infectious Virus ◽  
Allantoic Fluid ◽  
Progressive Reduction ◽  
Virus Particles ◽  
Host Virus Interactions ◽  
Virus Interactions ◽  
Available Information ◽  
Virus System

An experimental analysis is here presented of the conditions that lead to the appearance of non-infectious hemagglutinins (NIHA) in the allantoic fluid of chick embryos injected with standard influenza virus (PR8 strain) which had been exposed to 37°C. in vitro for various periods of time. On progressive reduction of the infectivity of the undiluted inocula from about 109 to 103 ID50 (103.2 HA units) the yields of infectious virus in 24 hours decreased in straight correspondence 1 millionfold, but those of hemagglutinins only by a factor of 10. Thus the proportions of NIHA in the yields increased sharply but the total quantity obtained decreased gradually. The quantities of infectious virus produced per ID50 injected were the same throughout this range; i.e., between 50 and 100 ID50, regardless of increasing proportions of heat-inactivated virus in the seeds. This value agrees with previous estimates of yields under other conditions. Thus, initiation and completion of first cycles by the infectious virus remaining in the inocula were not, or at most, slightly inhibited. The inactivated virus, therefore, failed to establish immediate interference. It was capable, however, of holding the infectious process to one cycle. Upon 10-fold dilution of the seeds essentially similar results were obtained except that a slight loss in interfering activity could now be detected with an increase in exposure to 37°C. With further dilutions little or no interference was noted. The capacity to yield NIHA decreased slowly during exposure of the seeds to 37°C. over a period of 5 days, thereafter more rapidly. It could not be restored by addition of infectious virus. Furthermore, since NIHA was obtained when the seeds contained as little as 102 or 103 ID50, it is unlikely that it was derived from those cells which had adsorbed both infectious and inactivated seed virus. It is suggestive that multiple adsorption of inactivated virus particles per se will yield NIHA. The available information, as discussed, favors the view that the NIHA does not represent seed virus in some form but is newly produced.

scholarly journals STUDIES ON HOST-VIRUS INTERACTIONS IN THE CHICK EMBRYO-INFLUENZA VIRUS SYSTEM

1956 ◽  
Vol 103 (6) ◽  
pp. 777-797 ◽  
Author(s):  
Oscar C. Liu ◽  
Kurt Paucker ◽  
Werner Henle
Keyword(s):  
Influenza Virus ◽  
Chick Embryo ◽  
Virus Production ◽  
Multiple Infection ◽  
In Ovo ◽  
Cell Receptors ◽  
The Media ◽  
Host Virus Interactions ◽  
Virus Interactions

Certain aspects of the formation of non-infectious hemagglutinins (NIHA) in the chick embryo infected with influenza virus have been analyzed. It was shown by the use of combined in ovo-deembryonation technics that little or no NIHA is released following infection with small doses of standard virus during the most active and constant growth periods of the virus extending to about the 36th hour of incubation in spite of the fact that multiple infection of cells must have taken place in the latter half of that period. A slight decrease in the ID50/HA ratios of the yields obtained after the 36th hour, coinciding with the falling off of virus production and release may possibly be explained in terms of inactivation of completed virus or leakage of as yet incompleted virus from damaged cells. Exposure of the entodermal cells of the allantois of eggs deembryonated shortiy after injection of saturation or near saturation inocula of standard seed to large quantities of infectious virus added to the media at various times after infection and not extending over more than 2 hours resulted in a decrease of the ID50/HA ratios of the progenies only during the first 2 or possibly 4 hours after the primary inoculation. Later addition did not influence the yields. As discussed, such sudden and heavy exposures of cells are not expected to occur during the infectious process induced by small inocula of standard seed. The possible role of destruction of cell receptors in NIHA production has been analyzed in several ways. The addition of receptor-destroying enzyme (RDE) to the media of deembryonated eggs after near saturation inocula of standard seeds, if anything, increased the ID50/HA ratios of the progenies, and that only when added during the first few hours following infection, presumably by reducing the changes for high multiplicity of infection of cells. In contrast, ultraviolet-inactivated virus, which retains its enzymatic activity, lowered, if anything, the ID50/HA ratios of the progenies, when present in the media of deembryonated eggs from the 2nd to 4th or possibly 6th hour after infection. Excessive amounts of irradiated virus may still cause some degree of interference under these conditions. Later addition of irradiated viruses were without effect with respect to NIHA production or interference. In attempts to alter the cell receptors prior to infection by potassium periodate (KIO4), it was noted that the addition of glycerol led to the appearance and partial retention for at least 24 hours of substances in the allantoic fluids which were capable of inactivating considerable proportions of standard virus. These data indicate that destruction of external cell receptors plays little if any role in NIHA production. The implications of these findings are discussed.

scholarly journals STUDIES ON HOST-VIRUS INTERACTIONS IN THE CHICK EMBRYO-INFLUENZA VIRUS SYSTEM

1949 ◽  
Vol 90 (1) ◽  
pp. 1-11 ◽  
Author(s):  
Werner Henle
Keyword(s):  
Chick Embryo ◽  
Influenza A ◽  
Allantoic Fluid ◽  
Active Virus ◽  
B Virus ◽  
The Difference ◽  
Host Virus Interactions ◽  
Virus Interactions ◽  
Allantoic Cavity ◽  
Partial Success

Upon injection of active influenza A or B virus into the allantoic cavity of the developing chick embryo, an average of only 70 per cent of the agent was adsorbed onto the tissue, as measured by the difference between the quantity of virus injected and that found free in the allantoic fluid of the injected eggs during the constant period. The degree of adsorption was similar, regardless of whether 109 or 102 ID50 of active virus was injected. Attempts to demonstrate the adsorbed virus in suspensions of the infected tissue met with partial success only in that not more than 1 to 5 per cent of the amount calculated to be adsorbed was actually found. All efforts to increase the yield of virus have failed. These results led to the suggestion that the seed virus, which participates in the propagation, becomes altered in such a way that it no longer may be demonstrated by infectivity titrations, whereas the active virus found represents superficially adsorbed virus, which does not multiply.

scholarly journals STUDIES ON HOST-VIRUS INTERACTIONS IN THE CHICK EMBRYO-INFLUENZA VIRUS SYSTEM

1951 ◽  
Vol 94 (4) ◽  
pp. 269-289 ◽  
Author(s):  
Oscar C. Liu ◽  
Werner Henle
Keyword(s):  
Influenza Virus ◽  
Growth Curve ◽  
High Speed ◽  
Allantoic Fluid ◽  
In Vitro Tests ◽  
Host Virus Interactions ◽  
Virus Interactions ◽  
Virus System

The role of inhibitors of hemagglutination in the evaluation of host-virus interactions in the chick embryo-influenza virus system has been analyzed. Comparisons were made between materials (allantoic fluids and membrane suspensions) derived from in vivo (growth curve) experiments at hourly intervals after inoculation, and from in vitro tests in which normal allantoic fluids and membrane suspensions were incubated with virus at 37°C. for various periods of time. In both instances large amounts of virus were added to the systems, resulting in comparable concentrations of the agent. The seeds employed were either fully active or irradiated by ultraviolet light to the extent that the virus lost its capacity to increase but kept its interfering and hemagglutinating properties. The various materials were assayed for (a) the hemagglutinating titers of the virus present in the systems before and after heating to 56°C.; (b) the concentration of inhibitor in the materials at various stages of incubation after heating to 70°C. for 30 minutes as measured by the hemagglutination-inhibition reaction with native or heated test virus (30 minutes 56°C.); and (c) the degree of adsorption of the hemagglutinins present in the materials onto chicken red cells at 0°C. and their subsequent elution at 37°C. The effects of receptor-destroying enzyme (RDE), treatment with sodium periodate, or high speed centrifugation on the inhibitory activities were studied in some of the tests. The essential results which indicate certain sources of error in the evaluation of host-virus interactions as well as means for studying virus activity at the early stages of the infectious process, were as follows: 1. Though some inhibitory effects on hemagglutination were noticeable in the allantoic fluid during the 1st hour after inoculation they were, as a rule, no longer apparent after this interval, and treatment with RDE did not increase the hemagglutinin titers. Thus, the interpretation of growth curve data concerning allantoic fluids hardly seems to be affected by inhibitor. On the other hand, striking effects were noted with the membrane suspensions of growth curve experiments in that RDE shortened the latent period to 2 hours and the titers in the first few positive samples (4 to 5 hours) increased) whereas in later harvests no such effect was noted. Under these conditions complement-fixation antigens and hemagglutinins made their appearance in the tissues simultaneously and not as previously reported the former prior to the latter. However, the infectivity showed increments only several hours after these two activities had become measurable. Thus the hypothesis of the stagewise development of influenza virus is still supported by these data. 2. Using the inhibition of hemagglutination technic it was found that the inhibitor in allantoic fluid rapidly decreased as a result of the action of active and irradiated virus, but destruction was never complete. In the membranes of the in vivo series only active seed led to loss of inhibitor, again without complete destruction, beginning at the time complement-fixing antigen and hemagglutinins became measurable. Irradiated seed was without effect in vivo whereas, in the in vitro tests it equalled the activity of the active virus. The implications of this difference in the effectiveness of active and irradiated seed in vivo with regard to the understanding of the mode of viral multiplication are discussed. 3. Although many factors may influence the shape of adsorption-elution curves it is felt that at 0°C. the extent of adsorption is directly related to the amount of inhibitor present in the systems. In the early hours after inoculation the degree of adsorption was relatively small but it increased gradually with the time of incubation. The inhibitor of adsorption was destroyed by RDE and NaIO4 and was only partially sedimentable by high speed centrifugation. In every respect studied its properties corresponded with the findings obtained with inhibitors in the hemagglutination-inhibition technic. Although the difference in the rapidity of inhibitor destruction as measured by the various technics might suggest a multiplicity of inhibitors it is felt that it rather denotes a greater sensitivity of the adsorption technic as compared to the others.

scholarly journals STUDIES ON HOST-VIRUS INTERACTIONS IN THE CHICK EMBRYO-INFLUENZA VIRUS SYSTEM

1954 ◽  
Vol 100 (1) ◽  
pp. 53-70 ◽  
Author(s):  
Werner Henle ◽  
Oscar C. Liu ◽  
Norman B. Finter ◽  
Keyword(s):  
Influenza Virus ◽  
Chick Embryo ◽  
Growth Curves ◽  
Virus Concentration ◽  
Continuous Exposure ◽  
Differential Growth ◽  
Thin Sections ◽  
Host Virus Interactions ◽  
Virus Interactions ◽  
Infectious Cycle

The period and rate of liberation of influenza virus from entodermal cells of the allantois have been studied by deembryonating eggs within a few minutes after infection, exchanging the medium thereafter at hourly intervals and assaying the virus concentration in the harvests thus obtained (differential growth curves). If the inoculum was sufficiently large, presumably all available cells immediately became infected and only 1 infectious cycle was expected to occur. If the inoculum was small, so that only a fraction of the cells adsorbed virus, the infectious process was held to 1 cycle by continuous exposure of the remaining susceptible cells to RDE. In either case, the results obtained indicate that once cells have been infected they produce and liberate virus at nearly constant rates for periods of 30 hours or longer before the yields decrease rapidly. Evidence has been presented which strongly suggests that such prolonged periods of liberation are observed not only in deembryonated eggs but also in the intact chick embryo. Attempts have been made in the discussion to reconcile these findings with previous estimates of the liberation period and to integrate them with histologic observations and electron micrographs of thin sections of infected allantoic membranes having a bearing on the mode of liberation.

scholarly journals STUDIES ON HOST-VIRUS INTERACTIONS IN THE CHICK EMBRYO-INFLUENZA VIRUS SYSTEM

1951 ◽  
Vol 94 (4) ◽  
pp. 305-322 ◽  
Author(s):  
Werner Henle ◽  
Oscar C. Liu
Keyword(s):  
Chick Embryo ◽  
Virus Particle ◽  
Prolonged Exposure ◽  
Influenza Viruses ◽  
Infective Virus ◽  
Virus Particles ◽  
E Coli ◽  
Partial Inactivation ◽  
Host Virus Interactions ◽  
Virus Interactions

Evidence has been presented that influenza viruses both of type A and B partially inactivated by ultraviolet irradiation may regain their capacity to propagate in the allantoic membrane of the chick embryo. In using such irradiated preparations as inocula for growth curve experiments it could be shown that the development of hemagglutinins as well as of infectivity preceded at rates resembling those noted with more than 10 times the amount of infective virus actually found in the irradiated seed. Partial inactivation of the inocula by heating to 56°C. gave similar results. The phenomenon was observed only with seed irradiated for short periods of time so that the virus particles sustained only few hits of radiation. On prolonged exposure resulting in numerous hits per virus particle the capacity of reactivation was lost. Likewise, an irradiated preparation capable of reactivation in the allantoic membrane, could not be diluted more than about 30-fold and still clearly produce this phenomenon. This indicated that reactivation is obtained only when one host cell adsorbs more than one non-infective virus particle but not upon adsorption of a single particle. These data are in striking agreement with the phenomenon of "multiplicity reactivation" observed in the bacteriophage-E. coli system by Luria and Dulbecco.

scholarly journals STUDIES ON HOST-VIRUS INTERACTIONS IN THE CHICK EMBRYO-INFLUENZA VIRUS SYSTEM

1949 ◽  
Vol 90 (1) ◽  
pp. 23-37 ◽  
Author(s):  
Werner Henle ◽  
Gertrude Henle
Keyword(s):  
Influenza A ◽  
Allantoic Fluid ◽  
Experimental Period ◽  
Virus Antigen ◽  
Immune Serum ◽  
Host Virus Interactions ◽  
Virus Interactions ◽  
Infectivity Titer ◽  
Infectious Cycle ◽  
Virus System

In agreement with earlier observations the infectivity titer in the allantoic fluids of chick embryos injected with influenza A virus remains constant for 5 to 6 hours before an increase in this activity can be noted. In contrast, the titers of hemagglutinin and complement-fixing antigen (virus antigen) have already begun to rise after 3 hours. The origin of the hemagglutinating and complement-fixing but non-infectious material is still obscure. In the allantoic membrane development of both the soluble and virus antigens can be demonstrated after the 2nd hour of incubation and 1 hour prior to an increase in hemagglutinins and 2 hours prior to a rise in infectivity. Thus there remain the first 2 hours during which no record of virus activity in the tissues can be obtained. Similar relationships are noted on dilution of the seed both in the allantoic fluids and membranes as long as the various properties reach measurable levels during the experimental period of one infectious cycle. Injection of high titered immune serum following infection with about 109 ID50 reduces the amount of demonstrable seed virus in the allantoic fluid and membrane without significantly affecting propagation of the agent in the tissues as measured by infectivity titrations. The production of hemagglutinins appears markedly reduced under these conditions whereas formation of complement-fixing antigen is only slightly delayed and decreased. No definite explanations for the various discrepancies between the infectivity and hemagglutination can be given at present.

scholarly journals STUDIES ON HOST-VIRUS INTERACTIONS IN THE CHICK EMBRYO-INFLUENZA VIRUS SYSTEM

1955 ◽  
Vol 101 (5) ◽  
pp. 479-492 ◽  
Author(s):  
Kurt Paucker ◽  
Werner Henle
Keyword(s):  
Influenza Virus ◽  
Infectious Virus ◽  
Growth Curves ◽  
Chick Embryos ◽  
Differential Growth ◽  
Host Virus Interactions ◽  
Virus Interactions ◽  
Allantoic Cavity

The role of inactivated influenza virus in the von Magnus phenomenon has been studied by exposing standard virus preparations in vitro to 37°C. for periods up to 6 or more days. The rate of inactivation of the infectious property of the line of PR8 virus employed was found to be approximately 1.1 log10 unit per day, denoting a half-life of 6½ hours. The rate of inactivation was similar in the allantoic cavity of chick embryos. On allantoic passage of such heated seeds without dilution it was seen that with a decrease in the infectivity of the inocula proportionately less infectious virus was found in the harvests. The yields of hemagglutinins were much less affected, and thus ID50/HA ratios of as low as 101.0 were observed in the 24 hour harvests. The ratios of the yields always equalled or were higher than those of the inocula. On 100- or 1000-fold dilution of the heated seeds standard virus was obtained. Growth curves in intact chick embryos or in deembryonated eggs (differential yields) revealed that non-infectious hemagglutinins appeared in the tissues or were liberated therefrom as soon as any virus activity became detectable. Furthermore, once maximal liberation had been established, infectious virus and non-infectious hemagglutinins were released for extended periods of time at nearly constant rates and in unchanging proportions, the latter depending upon the seed employed. Heated standard virus and undiluted passage seeds (von Magnus), selected on the basis of similar ID50 and HA concentrations, failed to yield similar results in differential growth curves in deembryonated eggs. Although the hemagglutinin titers in the 2-hourly harvests were nearly identical, the undiluted passage seeds produced as little as 1 per cent of the infectious virus which was derived from the heated inocula. Thus considerable differences exist between the 2 types of seeds.

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