Influenza virus and its mucoprotein substrate in the chorioallantoic membrane of the chick embryo. II. Stepwise inactivation of substrate and its relation to the mode of viral multiplication

The fate of heat-stable inhibitors (HI) of hemagglutination by influenza viruses in the infected chorioallantoic membrane has been studied. The amount of soluble HI extractable from the normal CAM is taken as a measure of the total mucoprotein substrate derived from the allantoic cells. It has been shown that a decrease in HI is demonstrable when all or nearly all cells of the allantois are involved in the infectious process. Once this condition is fulfilled, viral multiplication in the CAM is associated with stepwise breakdown and partial restoration of HI. The periodicity of these steps is in close agreement with earlier reports by others on the duration of primary and secondary cycles of viral multiplication. Periodicity of viral multiplication and of HI breakdown with subsequent restoration is particularly pronounced in eggs pretreated with metaperiodate or with mixtures of periodate and glucose or glycerol. The stepwise nature of the HI breakdown indicates that it is in some way related to intracellular phases of viral reproduction, and that individual cells produce virus in cycles rather than continuously. A possible mechanism of this process is proposed.     

Studies on host-virus interactions in the chick embryo-influenza virus system. IV. The role of inhibitors of hemagglutination in the evaluation of viral multiplication

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.     

Studies on host-virus interactions in the chick embryo-influenza virus system

The usefulness of the deembryonation technic has been analyzed as a tool in the study of various problems in the growth cycle of influenza virus in the entodermal cells of the allantoic of chick embryos. Various improvements in the deembryonation technic have been described. The method readily permits repeated sampling of the medium at various stages after infection (cumulative growth curves) or frequent exchanges of the medium (differential growth curve). However, the yield of infectious virus or of hemagglutinins is less than that observed in the intact chick embryo. The difference observed is greater than can be accounted for by the reduction in the available host cells and is assumed, therefore, to be due in part to interruption of blood and nutrient supply to the cells. This handicap can be overcome by the combined in ovo-deembryonation technic, in which deembryonation is performed at any desired time after infection of the intact chick embryo, and the medium is collected and analyzed after 1 to 3 hours of further incubation. The value of the technic is demonstrated by the fact that liberation of virus from infected cells can be detected earlier than in the intact egg. Furthermore, it continues at a nearly constant rate for many hours, thus proving to be erroneous previous inference which had been based upon in ovo experiments. The technic also permits readily the addition and subsequent removal of substances that might interfere with viral propagation. As an example a study was made of the effect of the receptor-destroying enzyme of V. cholerae (RDE) when added to the medium of eggs infected prior to deembryonation. By carefully grading the dose of virus and using an appropriate amount of RDE, one-step growth curves were obtained indicating that those cells not directly invaded by the seed virus were subsequently protected against infection by action of the enzyme. The smaller the amount of virus the less RDE was required in order to note a protective effect. With a decrease in the period of exposure to RDE regeneration of cell receptors became increasingly more apparent in that correspondingly greater amounts of virus were produced and liberated late in the incubation periods. These results confirmed and extended those reported by Stone. More extensive applications of these technics will be reported in subsequent papers of this series.     

Reactions between influenza virus and a component of allantoic fluid

Evidence is presented which shows that there is a component present in normal allantoic fluid, probably mucoprotein in nature, capable of combining with influenza A virus (PR8), and that following combination between this component and the virus only partial dissociation of the complex occurs. Evidence is also presented which strongly suggests that the component is present in virus-infected allantoic fluid in which it is in part combined with the virus and in part free although altered by viral action. The probability that the component is present as well in highly purified preparations of influenza virus, and its effect upon various reactions obtained with this agent are discussed.     

Studies on host-virus interactions in the chick embryo-influenza virus system. IX. The period of liberation of virus from infected cells

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.     

THE DEMONSTRATION OF ONE-STEP GROWTH CURVES OF INFLUENZA VIRUSES THROUGH THE BLOCKING EFFECT OF IRRADIATED VIRUS ON FURTHER INFECTION

After allantoic injection of chick embryos with a known amount of influenza virus, the process of adsorption of the agent onto host cells and infection of them can be interrupted at a given time by the administration of large quantities of heterologous virus inactivated by irradiation. A sudden great increase in the amount of free virus in the allantoic fluid occurring after 6 hours in the case of the PR8 strain, and 9 hours in that of the Lee strain, indicates that the untreated virus associated with the host cells has multiplied. The length of the period preliminary to this increase remains the same even though the concentration of the original inoculum is varied over a wide range. Since administration of the irradiated virus leaves no susceptible host cells, because of the interference phenomenon, and further adsorption of active virus is minimized or entirely prevented, practically the entire new increment of virus can be found in the allantoic fluid and assayed; for every ID₅₀ adsorbed about 50 ID₅₀ are released. Homologous irradiated virus, on the other hand, when injected after infection of the allantoic sac, reduces the yield of virus to a more or less considerable extent. Some inhibitory effect can still be observed when the homologous irradiated virus is given several hours after infection. This effect is linked to the virus particle and destroyed by prolonged irradiation.     

Growth characteristics of influenza virus concerning the binding of virus by host cells

Under certain conditions, influenza virus may bind to chorioallantoic membrane and the infectious property is retained upon prolonged incubation of the complex. Apparently the bound active virus is not functioning in the initiation of viral increase. The bound infectious virus may be partially removed by extensive washing. The characteristics of the washing are suggestive of a reversible equilibrium type of binding. Binding will also occur when the tissue has been pretreated with RDE or in the presence of AMPS. However, under these conditions the binding is of a lesser degree. When the tissue has been treated with RDE and AMPS is present, no stable binding occurs. In the presence of AMPS, the initiating activity is bound but cannot function in promoting viral increase. It is proposed that active virus is held by two types of binding at the same site; one type of binding is sensitive to the action of RDE; the second type is sensitive to the blocking effect of AMPS. Virus can be held to the receptor site by either type of binding or both. It is further suggested that the bound infectious virus is a result of an abortive attempt at initiating infection. The nature of the binding of infectious virus is of significance for understanding the binding of initiating activity.     

Engineering substrate promiscuity in 2,4-dichlorophenol hydroxylase by in silico design

2,4-Dichlorophenol hydroxylase (2,4-DCP hydroxylase) is a key enzyme in the degradation of 2,4-dichlorophenoxyacetic acid in the hydroxylation step in many bacteria. Our previous study demonstrated that a 2,4-DCP hydroxylase (TfdB-JLU) exhibits broad substrate specificity for chlorophenols (CPs) and their homologues. In this study, TfdB-JLU has been engineered by rational design to further broaden its substrate scope towards CPs. We dissect the architectures of enzymes from oxidoreductase families to discover their underlying structural sources of substrate promiscuity. A homology model of TfdB-JLU has been built and docking experiments of this homology model with its natural substrate 2,4-DCP reveal that the phenyl rings of 2,4-DCP form strong interactions with residues His47, Ile48, Trp222, Pro316, and Phe424. These residues are found to be important for substrate binding in the active site. Then, the site-directed mutagenesis strategy has been applied for redesigning substrate promiscuity in TfdB-JLU. The TfdB-JLU-P316Q variant obtained shows a significant enhancement of activity (up to 3.4-fold) toward 10 CP congeners compared to wild-type TfdB-JLU. Interestingly, the active improvements of TfdB-JLU-P316Q toward CP congeners show significant difference, especially for active improvements of positional congeners such as 3-CP (1.1-fold) compared to 4-CP (3.0-fold), as well as 2,3-DCP (1.2-fold) compared to 2,5-DCP (3.4-fold). Structural analysis results indicate that the improvement in substrate promiscuity of the variant enzyme compared to the wild-type enzyme is possibly due to the increase of non-bonding interaction. The results suggest that exploiting enzyme–substrate promiscuity is promising, which would provide a starting point for designing and engineering novel biological catalysts for pollution removal.

In silico designed 2,4-DCP hydroxylase exhibits broader substrate promiscuity for chlorophenols than that of the wild-type enzyme.     

STUDIES OF TWO KINDS OF VIRUS PARTICLES WHICH COMPRISE INFLUENZA A2 VIRUS STRAINS : I. CHARACTERIZATION OF STABLE HOMOGENEOUS SUBSTRAINS IN REACTIONS WITH SPECIFIC ANTIBODY, MUCOPROTEIN INHIBITORS, AND ERYTHROCYTES

Two kinds of virus particles have been found in varying proportions in influenza A2 strains isolated during the 1957 pandemic. Pure populations of the different particles were obtained, and these substrains were genetically stable on serial passage in the chick embryo. The two virus particles differ markedly in several biological properties though they are antigenically similar. One kind of particle, designated "+," is relatively sensitive to specific antibody, is highly sensitive to inhibition by serum inhibitors and urinary mucoprotein, fails to elute or elutes very slowly from human erythrocytes, and is capable of agglutinating erythrocytes treated extensively with V. cholerae filtrate. The other particle, designated "-," is relatively insensitive to antibodies and urinary mucoprotein, completely insensitive to serum inhibitors, elutes rapidly from erythrocytes, and can agglutinate erythrocytes treated extensively with V. cholerae filtrate. Both "+" and "-" particles destroy virus receptors on urinary mucoprotein. The relative proportions of these two particles determine the characteristics of parent strains in reactions with specific antibody, mucoprotein inhibitors, and erythrocytes. The "+" and "-" particles with several easily identifiable markers are well suited for genetic studies.     

The effect of polysaccharides on the reaction between erythrocytes and viruses,with particular reference to mumps virus

Polysaccharides which cause inhibition of the multiplication of mumps virus in the allantoic sac may or may not cause inhibition of hemagglutination by the virus. Moreover, such substances may or may not prevent adsorption of the virus by erythrocytes. The available evidence indicates that polysaccharides active as inhibitors do not block adsorption of mumps virus by cells of the living allantoic membrane. With influenza A, influenza B, and Newcastle disease viruses, as well as with PVM, there also appears to be a lack of correlation between the in vitro and in vivo inhibiting activity of polysaccharides.     

An immunofluorescence assay for double-stranded DNA antibodies using the Crithidia luciliae kinetoplast as a double-stranded DNA substrate.

Circulating antibodies to dsDNA are found predominately in SLE and are seen most often in those patients with active systemic disease, particularly severe lupus glomerulonephritis. An IIF technique for measuring these antibodies has recently been described. This uncomplicated assay employs the kinetoplast of the nonpathogenic hemoflagellate Crithidia lucilliae as a dsDNA substrate. We herein report our experience with this assay, emphasizing the methodology and interpretation of results. Although slightly less sensitive than a radioimmunoassay, we find that this IF technique is a specific and reliable qualitative method for detecting anti-dsDNA. An estimate of the amount of DNA antibody present can be obtained by serum titration. The test was positive in only two patient groups tested, SLE (48%) and MCTD (20%).     

Studies on the psittacosislymphogranuloma group. III. The effect of aureomycin on the propagation of virus in the chick embryo

The findings presented indicate that aureomycin could become associated with tissue of the chick embryo by both hematogenous distribution and direct adsorption. Treatment of chick embryos infected with MP virus with 1 mg. of aureomycin by the allantoic route caused an inhibition of virus growth in the allantoic membrane. The drug had no effect on "inert" virus, and appeared to have little effect on adsorption of virus to host tissue. Complete inhibition of growth during the time interval corresponding to the first cycle of multiplication could be achieved only if the drug was administered within 6 to 8 hours after virus inoculation. Partial inhibition of virus multiplication could be achieved even if the administration was delayed as late as 24 hours after infection. In these experiments the chief role of the antibiotic appeared to be one of virustasis reflected in a prolongation of the latent period (non-infectious phase). The virus was able to resume its growth when a critical low level of the drug in the allantoic membrane was reached. When infectivity titrations were carried out using various tissues and organs of treated and untreated embryos, it was found that no virus was detectable in the brains of treated embryos as late as 192 hours after inoculation of virus. This was in contrast with the findings in allantoic membranes and livers of such embryos; these organs showed virus at 120 and 144 hours, respectively. In untreated controls, virus appeared in membranes at 24 hours, in the liver at 48 hours, and in the brain at 72 hours.     

Ge nanowires on top of a Ge substrate for applications in anodes of Li and Na ion batteries: a first-principles study

In this study, density functional theory (DFT) was used to research the adsorption and diffusion features of Li and Na on Ge nanowires on top of a Ge substrate. The adsorption energies at different positions are 0.71–1.28 eV for Na and −2.96–−2.13 eV for Li. The adsorption energies can be further reduced by surface modification with one or two P atoms. In particular, the sidewall of the Ge nanowire modified by two P atoms is most favorable to adsorb Li/Na. In addition, we used the nudged elastic band (NEB) method to study the diffusion pathways of Li/Na on the sidewall of Ge NW and the Ge substrate and computed their energy barriers. When Li or Na diffuses across the Ge NW, the energy barrier is 0.65 or 0.79 eV, indicating that the Ge NW can be applied to anodes in lithium and sodium ion batteries. Finally, the insertion of more lithium and sodium atoms into the Ge NW would cause volume expansion and the average length of Ge–Ge bonds to increase. This work will contribute to studying the adsorption and diffusion of Li and Na on nanowires with a substrate and the volume expansion caused by the insertion of Li/Na into the nanowires. Additionally, it provides guidance for designing Ge anodes for sodium ion batteries.

(a) The energy curves along the D–E pathway for the Li/Na diffusion; the side-view of the trajectories of (b) Li and (c) Na diffusion across the Ge NW.     

Effect of substrate size on immunoinhibition of amylase activity

Immunoinhibition assays are hypothesized to work by antibodies blocking substrate access to enzyme active sites. To test this hypothesis, the inhibition of amylase isoenzymes by monoclonal and polyclonal antisera was assessed using substrates of varying sizes: chromogenic sustrates 3, 5, or 7 glucose units in length, novel synthetic macromolecular substrates, and starch. The synthetic macromolecular substrates consisted of small oligosaccharide substrates linked to an inert polymer that conferred a large size to substrate molecules as determined by gel filtration chromatography. When substrate size increased, amylase activity could be inhibited equivalently by antibody concentrations that are 10-fold lower. Progressively less polyclonal serum was required to inhibit amylase activity as substrate length increased from 3 to 5 to 7 glucose units and as size was increased by linkage to a polymer. Different effects of substrate size were observed with two monoclonal antibodies. One monoclonal antibody blocked amylase activity independent of substrate size, while another monoclonal antibody had little inhibitory effect except using starch as substrate. We conclude that use of larger substrates can expand the repertoire of inhibitory epitopes on enzymes and convert a noninhibitory antibody into an inhibitory one.     

The assessment of acid-base disturbance in man by the use of carbon dioxide titration curves.

1. Carbon dioxide titration curves were determined in vivo in dog and man at various degrees of acute non-respiratory acidaemia and alkalaemia. 2. The slope of the CO2 titration curve (delta log Pco2/delta pH) was found to increase with the severity of the acute non-respiratory alkalaemia the slope (delta log Pco2/delta pH) tended towards unity. 3. A simple scheme based on the CO2 titration curves determined in vivo has been proposed for the assessment of acute acid-base disturbances in man. 4. Carbon dioxide titration curves were also determined in vivo in patients with chronic respiratory and non-respiratory acidaemia and it was found that these curves were not significantly different from those obtained in states of acute acid-base disturbances. It is therefore suggested that the scheme described in this paper is applicable to all acid-base disturbances.     

Morphological and quantitative comparison between infectious and non-infectious forms of influenza virus

Electron microscopic study has revealed the morphological entity responsible for the rise in viral hemagglutinin observed in brains of mice after intracerebral inoculation of non-neurotropic strains of influenza virus. This rise in hemagglutinin, although dependent on inoculation of fully infectious virus, is not associated with an increase in infectious titer. The hemagglutinating principle is functionally similar to the "incomplete" influenza virus which can be obtained from chick embryos by serial egg-to-egg transfer of undiluted, infected allantoic fluid according to the method of von Magnus. A method has been described which facilitates selective adsorption of viral particles recovered from organ extracts on saponine-lysed ghosts of fowl erythrocytes. This procedure has been utilized in studying the morphology of non-infectious, hemagglutinating virus from chorio-allantoic membranes or mouse brains and in comparing these two forms with each other and with ordinary, infectious (standard) influenza virus. Standard virus isolated from allantoic fluids or membranes of infected eggs was found to contain uniform particles of predominantly spherical shape with smooth surface and even density, resembling those described by others. The appearance of such particles was not affected by the procedure of extraction and concentration used. In contrast, non-infectious, hemagglutinating virus obtained either from allantoic sacs ("undiluted passages") or from mouse brain was pleomorphic and seemed to consist of disintegrating particles. The majority appeared flattened and bag-like and had a rough, granular surface and reduced, uneven density. 37 per cent of the non-infectious particles isolated from mouse brain infected with the non-neurotropic strain WS had diameters in excess of 170 mµ, as compared with only 2 per cent of the particles of the parent strain itself. Regardless of whether or not the contrast in appearance of standard and of non-infectious particles was due to differing resistance to the preparatory treatment, it indicated the existence of basic structural differences between the two types of virus. Correlation of particle counts with hemagglutinin titers has shown that the non-infectious virus obtained from mouse brain is, unit for unit, an equivalent counterpart of standard virus derived from infected eggs. The end-point of hemagglutination in a pattern test corresponds for both forms to that dilution at which the ratio virus particles/red cells approaches one. The quantitative data based on particle counts support the assumption that non-infectious virus arises in mouse brain as a product of viral multiplication.     

Binding parameters and thermodynamics of the interaction of imino sugars with a recombinant human acid alpha-glucosidase (alglucosidase alfa): insight into the complex formation mechanism

BACKGROUND: Recently, enzyme enhancement therapy (EET) for Pompe disease involving imino sugars, which act as potential inhibitors of acid alpha-glucosidases in vitro, to improve the stability and/or transportation of mutant acid alpha-glucosidases in cells was studied and attracted interest. However, the mechanism underlying the molecular interaction between the imino sugars and the enzyme has not been clarified yet. METHODS: We examined the inhibitory and binding effects of four imino sugars on a recombinant human acid alpha-glucosidase, alglucosidase alfa, by means of inhibition assaying and isothermal titration calorimetry (ITC). Furthermore, we built structural models of complexes of the catalytic domain of the enzyme with the imino sugars bound to its active site by homology modeling, and examined the molecular interaction between them. RESULTS: All of the imino sugars examined exhibited a competitive inhibitory action against the enzyme, 1-deoxynojirimycin (DNJ) exhibiting the strongest action among them. ITC revealed that one compound molecule binds to one enzyme molecule and that DNJ most strongly binds to the enzyme among them. Structural analysis revealed that the active site of the enzyme is almost completely occupied by DNJ. CONCLUSION: These biochemical and structural analyses increased our understanding of the molecular interaction between a human acid alpha-glucosidase and imino sugars.     

STUDIES OF THE HEMOLYSIS OF RED BLOOD CELLS BY MUMPS VIRUS : II. THE RELATIONSHIPS OF HEMAGGLUTINATION, VIRUS ELUTION, AND HEMOLYSIS

The relationship between hemagglutination and hemolysis by the mumps virus has been studied under conditions which affect (a) the receptors of chicken red cells and (b) the adsorption and subsequent elution of the virus from these cells. The results show that the hemolytic action of the virus appears to involve some of the same receptor areas of erythrocytes that are implicated in hemagglutination. Materials such as allantoic fluid, egg white, and red cell extract, which inhibit the agglutination of chicken red cells by mumps virus, also interfere with its hemolytic activity. Of these inhibitors, egg white and red cell extract, which are readily destroyed by the virus during incubation at 37°C., exert a greater antagonistic effect on hemagglutination than on hemolysis. Heated mumps virus or unheated influenza virus interferes with the hemolysis of red cells by untreated mumps virus. Though hemolysis takes place during elution of the virus after its adsorption on the red cell, the processes are apparently distinct. The hemolytic activity is easily affected by certain conditions of pH and temperature which have no effect on the ability of mumps virus to adsorb on and elute from red cells.     

STUDIES ON THE NATURE OF THE VIRUS OF INFLUENZA : I. THE DISPERSION OF THE VIRUS OF INFLUENZA A IN TISSUE EMULSIONS AND IN EXTRA-EMBRYONIC FLUIDS OF THE CHICK

A considerable fraction of the influenza A virus contained in infected allantoic fluid of the developing chick is not sedimentable under conditions which remove virus activity almost completely from filtrates of emulsified mouse lung. The infectious unit from tissue suspensions is about 100 mµ in diameter and is of the same chemical composition as particles of the same size and abundance separated from normal tissues by an identical procedure. Evidence has been presented showing that the infectivity can be, and probably is, carried on such normal cell components as an adsorbate. Other non-infective particles such as erythrocytes may also become infectious units through adsorption of the virus. The virus occurs in allantoic fluid in two states of dispersion. A variable percentage is associated with particles considerably less than 100 mµ in diameter, probably more nearly 10 mµ, while the remainder is reversibly aggregated. Reversal to the more disperse state may be effected by dilution, sonic vibration, or moderate heat treatment.     

PRECIPITIN REACTIONS OF HIGHLY PURIFIED INFLUENZA VIRUSES AND RELATED MATERIALS

Antisera to purified PR8 virus, to purified protein from normal allantoic fluid, and to purified normal mouse lung particles were obtained from hyper-immunized rabbits and used in quantitative precipitin tests employing various purified preparations of influenza virus and related materials as antigens. The results of those tests indicated that the most highly purified preparations of PR8 or of Lee influenza virus obtained from infectious allantoic fluid contain an antigen characteristic of normal allantoic fluid and likewise that highly purified mouse lung PR8 virus contains an antigen characteristic of normal mouse lungs. Since the infectivity of virus preparations which were ultracentrifugally and electrochemically homogeneous was precipitated by the appropriate antisera to normal antigens, it was concluded that the normal antigens constitute a part of the 100 mµ particles with which influenza virus activity is at present deemed to be associated. It was estimated from quantitative precipitin data that the most highly purified preparations of PR8 and of Lee influenza viruses obtained from infectious allantoic fluid contain at least about 20 and 30 per cent, respectively, of an antigenic structure characteristic of the sedimentable protein of normal allantoic fluid.