Interferon

Alick lsaacs Notionol Institute for Medico1 Research. Mill Hill. London. England

I. Introduction . . . . . . . . . . . . . . . . A Definition . . . . . . . . . . . . . . . . B. Viral Inhibitory Substances Recovered from Virus Infections . . C Techniques of Assaying Interferon . . . . . . . . . I1. Production by Different Cells and Viruses . . . . . . . . A Production by Cells of Different Animal Species . . . . . B. Production by Different Varieties of Cells . . . . . . . C. Production of Interferon by Inactivated Virus . . . . . . D . Production of Interferon by Live Virus . . . . . . . . E. Senstivity of Different Viruses to the Antiviral Action of Interferon F. Factors Concerned with the Production of Interferon . . . . 111 Properties . . . . . . . . . . . . . . . . . A Physicochemical Properties . . . . . . . . . . . . B Biological Properties . . . . . . . . . . . . . IV Purification . . . . . . . . . . . . . . . . . V. Mode of Action . . . . . . . . . . . . . . . A. Site of Action in Virus Growth Cycle . . . . . . . . B. Conditions Required for Action of Interferon . . . . . . C. Mechanism of Action . . . . . . . . . . . . . D. Effects of Interferon on Cells . . . . . . . . . . . E . Production and Action of Interferon at the Cellular Level . . . VI Interferon and Recovery from Virus Infection . . . . . . . A Recovery from Virus Infection in Vitro . . . . . . . . B Recovery from Virus Infection in the Chick Embryo . . . . C. Recovery from Virus Infection in Adult Vertebrates . . . . D Virus Virulence . . . . . . . . . . . . . . . VII. The Role of Interferon in Normal Cells . . . . . . . . . VIII Interferon as a Possible Therapeutic Agent . . . . . . . . References . . . . . . . . . . . . . . . . .

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I INTRODUCTION

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A Definition Interferon is the name that was given to an antiviral substance produced by the cells of many vertebrates in response to virus infection . It appears to be of protein or polypeptide nature. it is antigenically distinct from virus. and it acts by conferring on cells resistance to the multiplication of a number of different viruses. 1

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B. Viral Inhibitory Substances Recooered from Virus Infections Interferon derived its name from virus interference, since it was first isolated and characterized during a study of this phenomenon (Isaacs and Lindenmann, 1957). However, similar substances were previously observed, although they were not characterized. @rskov and Andersen (1938) found that within a short time of the initiation of vaccinia1 infection of the rabbit skin local “antibody” could be detected at the site of infection at a time when none could be found in the serum. In retrospect, grskov (personal communication, 1962) feels that this was interferon, not antibody, Card (1944)studied tissue immunity in mouse encephalomyelitis and observed an interfering factor that was separable from the virus. He found that a suspension of brain from mice infected with Theiler’s mouse encephalomyelitis virus was able to inhibit the growth of virulent virus in fresh mice; the inhibitory factor seemed to be cell-bound and did not act by combining with the challenge virus. Lennette and Koprowski (1946) found that infected cultures of chick and mouse embryo tissue when freed of virus showed a very weak viral inhibitory action which they thought could not explain the viral interference they observed. Nagano and Kojima (1954) studied a similar experimental situation to that of grskov and Andersen (1938) and they also found a virus-inhibitory substance separable from the infecting virus in extracts of infected rabbit skin. However, these authors were unable to decide whether the inhibition found was an immunological or an interference effect; indeed it is difficult in experiments in animals to distinguish how much of the viral inhibitory action found might be due to specific antibody, to inactivated virus or viral antigens capable of inducing virus interference, or to interferon. Thus the recent experiments of Matumoto et al. (1959) show that infection of mice with neurotropic Rift Valley fever v i r u s protects them against the virulent pantropic variant; protection is slight if the two viruses are injected together but increases the longer the interval between the two. Their results suggest that viral interference may have played a more importarit role in inducing protection when the two viruses were injected together, but with lengthening interval of time between the two, specific immunity may have become more important. To distinguish these it has become necessary to carry out experiments in chick embryos or in tissue culture, in order to exclude antibody, and to allow adequate characterization of the virus inhibitory substances found. In this review, no attempt will be made to summarize studies on &US interference which have been thoroughly covered by Henle (1950), Schlesinger ( 1959), and Wagner (1960). Nor will any attempt be

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made to evaluate the role of interferon in virus interference beyond drawing attention to the evidence described by Henle et al. (1959) and Isaacs (1959) that many examples of virus interference can be accounted for by the production of interferon by cells in response to contact with the interfering virus. Although work on interferon began as an attempt to find an explanation of viral interference, an early observation was that once formed, interferon was rapidly liberated from cells and could be found in much higher concentration in the extracellular fluid than within cells (Isaacs and Lindenmann, 1957; VilEek, 1961; Bader, 1962). This raised the possibility that interferon was capable of protecting not only the cells initially infected but also neighboring cells. Thus, attention was soon directed toward considering the possible role of interferon in cellular resistance to virus infection, in general, and in the processes of recovery from virus infection in particular. These themes will therefore be dealt with in Section VI of this review in place of a consideration of virus interference.

C. Techniques of Assaying Znterferon Many different techniques are used to assay interferon. The most generally used, however, measure the degree of inhibition in the ability of treated cells to produce virus after infection. This is measured either as a diminution of the yield of virus from treated cells or as a diminution in the abililty of treated cells, when infected, to initiate the production of a viral lesion (e.g., a plaque in cell monolayers). The technique which was first used (Isaacs and Lindenmann, 1957) was to measure the reduction in the yield of influenza virus hemagglutinin from pieces of chick chorioallantoic membrane infected in vitro by the technique of Fulton and Armitage (1951). This method was based on the finding of a linear relationship between the degree of virus inhibition produced and the concentration of interferon used (Isaacs et al., 1957). This type of method has now been largely replaced by a plaque assay method in which the concentration of interferon that will produce a 50%reduction in the plaque count in a cell monolayer is measured (Wagner, 1960). In assays with Merent experimental systems a linear relation has been found between the degree of reduction of the plaque count and the logarithm of the concentration of interferon over quite a wide range of concentrations, so that the end point of the assay can, if necessary, be determined by interpolation. Gifford et a2. (1963) have developed an assay of this kind based on the method of Postlethwaite (1960) for producing plaques with vaccinia virus without using an agar overlay. When the logarithm of interferon concentration was plotted against the reduction in plaque count, an S-shaped curve was formed which

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was linear over a certain range of concentrations of interferon. They also found a linear relationship when the relative average plaque diameter or the total plaque area was plotted against the logarithm of interferon concentration. A third method of assay is based on the size of the zone of protection produced in a sheet of virus-infected cells when a cup containing interferon is placed over the agar overlay. Porterfield (1959) showed that there was a linear relationship between the concentration of interferon, plotted logarithmically, and the area of the protected zone. Another technique used is to measure the degree to which a culture of cells is protected against the cytopathic action of virus as judged microscopically ( Sellers and Fitzpatrick, 1962). This assay gives a linear relationship between the logarithm of the concentration of interferon and the logarithm of the amount of virus inhibited. Sueltenfuss and Pollard (1963) have developed a very sensitive assay which is based on inhibition of the development of the inclusions produced by psittacosis virus, as judged by fluorescence microscopy of cells stained with acridine orange. These are the basic methods most commonly used in assaying interferon; the review by Porterfield (1963) gives a more detailed description of the techniques used. 11. PRODUCTION BY DIFFERENT CELLSAND VmusEs Production of interferon was studied first in chick cells infected with inactivated influenza virus, but it soon became clear that similar substances were produced by the cells of many animal species in response to infection with a variety of different viruses.

A. Production by C e h of Different Animal Species Among the animal species whose cells have been shown to produce interferon in vitro are chickens, ducks, mice, rats, guinea pigs, hamsters, rabbits, ferrets, dogs, sheep, pigs, cows, monkeys, and man. Table I of the review by Ho (1962b) gives many references to work describing production of interferon by different cell-virus systems. Production during the course of infection in uivo has been demonstrated in chick embryos, mice, and rabbits, but has been much less studied than production in uitro. The fact that birds produce interferon raises the question of how early in evolution such a mechanism might have arisen. Virus interference has been found among bacterial and plant viruses but it is not known whether it is mediated by substances similar to interferon, although one report has appeared indicating that an interferon-like substance was produced by Pseudonumas aemgirwsa infected with

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bacteriophage (Mercer and Mills, 1960). The nature of the repressor that is responsible for some cases of immunity to superinfection shown by lysogenic bacteria (Jacob, 1959) is not yet known, but the fact that the immunity tends to be specific toward the infecting phage does not favor the suggestion that the repressor might function in the same way as interferon.

B. Production by Diferent Varieties of Cells No systematic study has been made of the production of interferon by cells from different organs, but no striking differences in the behavior of cells have been found in in uitro or in uiuo studies. Thus, in uiuo, production of interferon has been observed in the mouse brain and lungs and in the rabbit skin, and in uitro, in chick chorionic and allantoic cells, human amnion cells, calf, dog, monkey, and human kidney cells, human thyroid cells, and human leucocytes. Until now, no differences have been observed between the behavior of epithelial cells or fibroblasts. Certain lines of tumor cells were thought at first to be poor producers of interferon (e.g., Henle et al., 1959) but this may be due to the fact that many tumor cell lines are very insensitive to the antiviral action of interferon, even to that produced in the same cells. Thus, Ho and Enders (1959a,b) found that HeLa cells produced interferon which they could assay on primary human amnion cells but not in HeLa cells. Similar findings were reported for KB cells by Chany (1961), for HeLa cells by VilEek ( 1962), and for a human amnion cell line by Mayer (1962).However, this is not an invariable finding since Cantell ( 1981a) and Isaacs et al. (1961b) have found that certain lines of HeLa cells show some sensitivity to the action of interferon, although less than that of primary human thyroid cells, in the case of one cell line studied. It was suggested by Isaacs et al. (1961b) that this behavior of tumor cells might reflect metabolic differences from normal cells, and it would be interesting to study this question in lines of HeLa cells differing in sensitivity to interferon. Embryonic cells have been used extensively to produce interferon, but chorioallantoic cells of 6-day chick embryos were found to produce only about one-tenth as much interferon as the cells of ll-day embryos after treatment with irradiated influenza virus ( Isaacs and Baron, 1980). Also suckling mice infected intranasally during the first day of life with parainfluenza 1 (Sendai) virus produced more virus but less interferon than did 4-week old mice similarly infected (Sawicki, 1961). The question of whether it might generally be found that cells show

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increased production of interferon with aging of the animal of their origin, or aging f n vitro, requires further investigation.

C. Production of Interferon by Znuctivated Virus First studies of interferon were carried out with inactivated myxoviruses. Among the viruses shown to produce interferon were influenza A and B, Newcastle disease, and fowl plague viruses, inactivated by irradiation with ultraviolet (UV)light, heating at 5 6 O or at 37OC., but not by treatment with formaldehyde (Burke and Isaacs, 1958b). Other viruses shown to induce the production of interferon when used inactivated are mumps (Cantell, 1961a), Rous sarcoma virus (Bader, 1962), vaccinia ( Glasgow and Habel, 1962), and herpes simplex (Waddell, 1962). Incomplete influenza virus, produced by repeated passage at high virus concentration, has been shown to induce interference (von Magnus, 1954) and to induce production of interferon when inoculated on the chick chorion at a site where virus multiplication does not occur ( Burke and Isaacs, l958a). Interferon induced by different viruses shows no evidence of specificity, i.e., it is not most active when tested against the homologous virus (Lindenmann et al., 1957). Ho and Breinig (1962) have found that Sindbis virus heated at 56OC. for 4 hours did not induce production of interferon but was able to “sensitize” cells so that they now produced interferon when infected with live Sindbis virus. A number of reports have appeared indicating absence of interferon production by arboviruses and enteroviruses when used inactivated (e.g., Ho and Enders, 1959b). With one arbovirus, inactivation by deoxycholate was found to produce a virus still capable of inducing interference but no interferon could be detected (Henderson and Taylor, 1961). However, the fact that interferon was not detected makes it difficult to conclude that none was produced since the conventional tests measure only excess interferon liberated from cells. Before concluding that a virus once inactivated does not produce interferon it will be necessary to examine different types of inactivation, since it is known that if influenza virus is heated too much (Isaacs and Lindenmann, 1957) or over-irradiated (Burke and Isaacs, 1958a), it loses its ability to produce interferon. The results of Ho and Breinig suggest that, at least with one virus, prolonged heating may have reduced its ability to stimulate the production of good titers of extracellular interferon while retaining its ability to sensitize cells to respond to infection by live virus by producing interferon. Iduenza virus more gently inactivated by heat was able to induce production of interferon and was found to sensitize cells to respond to infection by live virus by producing a rapid synthesis of interferon (Burke and Isaacs, 1958b). Recently, VilEek (1963) has studied production of interferon in

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chick cells induced by tick-borne encephalitis virus inactivated by incubation for various periods of time at 37OC. He has concluded that interferon production could be demonstrated only when live virus was present. VilEek points to the fact that among viruses that have been shown to induce production of interferon when used in the inactivated form it has not yet proved possible to obtain infective viral nucleic acid. Alternatively, in the viruses among which, until now, no clear evidence of production of interferon by inactivated viruses has so far been shown, it is readily possible to prepare infective viral RNA (ribonucleic acid). This seems to be an interesting division among viruses, although so far its significance is unknown.* The findings quoted above concern production of interferon by virus which has been rendered noninfective by a particular treatment. The converse situation is infection by live virus of cells that are “insusceptible,” implying that the cells are unable to support a complete cycle of growth by a particular virus. Interferon production of this kind has been found with influenza virus in chick chorionic cells (Lindenmann et al., 1957) and by parainfluenza 1 and measles viruses in human leucocytes (Gresser, 1961b). It seems clear, therefore, that virus multiplication is not essential for production of interferon. The question of which viral constituent stimulates cells to produce interferon will be discussed in Section VII.

D. Production of Interferon by Live Virus The term l i v e virus” is used to denote virus prepared in such a way as to avoid as much as possible any loss of infectivity. However, with animal viruses kept under optimal conditions, the majority of the virus particles are incapable of initiating infection, the ratio of infective particles to total virus particles being usually of the order of 1 to 10. Since some strains of influenza virus grown in suspended chick chorioallantoic membranes gave rise to good yields of interferon within 6-12 hours of infection with inactivated virus, and poorer yields of interferon at a later stage of infection with live virus, it is possible that production of interferon by live virus is due largely to particles in the virus population that are not undergoing multiplication. This question cannot be resolved until methods are available for measuring the yield of interferon from single cells. It is discussed further in Sections II,F and VI. The review by Ho (196213) gives in Table I a list of references to production of interferon by different live viruses. Viruses shown to

’However, Gifford and Heller (1963)have now found good yields of interferon on infecting chick cells with an arbovirus (Chikungunya virus) inactivated by incubation for 23 hours at 35°C.

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induce production of interferon include RNA and DNA (deoxyribonucleic acid) viruses, all ranges of size from foot-and-mouth disease virus (Dinter, 1960) to the pox viruses (Nagano and Kojima, 1958), cytolytic viruses, e.g., arboviruses, and tumor viruses, e.g., polyoma (Allison, 1961) . It seems justifiable to conclude, therefore, that production of interferon is a very general response of cells to virus infection. The yield of interferon differs greatly with different viruses grown in the same cells or with a single virus grown in different cells. This is discussed further in Section VI in relation to the problem of virus virulence.

E . Sensitivity of Diflerent Viruses to the Antiviral Action of Intsrferon In addition to the differences in the yield of interferon which they can induce, viruses also differ in their sensitivity to the antiviral action of interferon on cells. The two properties give the impression of being related, since it is frequently found that viruses that give good yields of interferon are sensitive to its antiviral action, and conversely, that viruses that give poor yields of interferon are generally much less sensitive to its antiviral action. It is not known whether there is any necessary relationship between these two properties. Possibly tests of the sensitivity of a virus to interferon measure indirectly the probability that a particle belonging to a particular virus population will induce the production of interferon instead of virus, in both normal cells and interferon-treated cells. Differences found in the sensitivity to the antiviral action of interferon may be quite considerable. Thus roughly 30 times more interferon was required to cause !%&inhibition of plaque production by Newcastle disease virus than by O’nyong-nyong virus grown in chick embryo fibroblasts ( Ruiz-Gomez and Isaacs, 1963a). An early observation was that herpes simplex virus was much more resistant to the action of interferon than vaccinia or cow pox viruses grown on the chick chorion (Isaacs et al., 1958). Ho and Enders ( 1959b) found that herpes simplex virus was much more resistant to interferon than vaccinia or Sindbis viruses grown in human amnion or human kidney cells. Relative resistance of herpes simplex was also observed by Cantell and Tommila (1960) in the rabbit cornea and by VilEek and Rada (1962) in chick embryonic cells, and the closely related pseudorabies virus was shown to behave similarly by VilEek (1962) and by Dinter and Philipson ( 1962). Adenovirus type 7 was found to be very resistant to the action of interferon in HeLa cells (Cantell, 1961a). Viruses that have been shown to be relatively resistant to the action of interferon include strains of fowl plague, Newcastle disease, herpes simplex, pseudo-

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rabies, and adenovirus. However, Glasgow and Habel (1962) found herpes simplex virus relatively sensitive to the action of interferon in mouse cells; it is not known whether this is due to the use of a different strain of virus, or different'cells from those used by other workers. As a general rule, vaccinia virus and many arboviruses and rhinoviruses seem to be relatively much more sensitive to the antiviral action of interferon (Baron et al., 1961; Sutton and Tyrrell, 1961) although differences in sensitivity among the arboviruses can be shown. Differences in sensitivity among different viruses have been related to differences in oxygen requirement, in optimal temperature for virus growth, and in virus virulence. These points are discussed in the following sections since they may throw some light on the mode of action of interferon.

F . Factors Concerned with the Production of Interferon Production of good yields of interferon was observed within 6 hours of infection of chick chorioallantoic membrane with heated influenza virus (Isaacs and Lindenmann, 1957) or infection of human leucocytes with parainfluenza 1 virus (Gresser, 1961b), which does not multiply in these cells. Incubation at about 37OC. was required, incubation at 2OC. giving no significant yield of interferon. Production of interferon continued for 24 hours when it gradually ceased, but a second inoculation of heated influenza virus at this time gave rise to a second crop of interferon ( Lindenmann et d.,1957). Irradiated influenza virus gave a more long-lasting stimulus, production of interferon being detectable in small amount even on the third day after infection (Burke and Isaacs, 195813). The fact that protein synthesis is required for the production of interferon is indicated by the inhibition of interferon formation produced by treating cells with p-fluorophenylalanine ( unpublished observations). Within a short time of being detected within cells interferon was rapidly liberated and was recovered in good yield from the suspending medium (Isaacs and Lindenmann, 1957). Interferon produced in chorionic cells could be shown to diffuse not only outward from the chorionic surface but also inward through the mesoderm to the allantoic cells (Isaacs et al., 1958).This rapid liberation from cells first suggested that interferon might be capable of protecting not only the cells initially infected but also neighboring cells. Early production and rapid liberation of interferon is characteristic of infection with inactivated or nonmultiplying virus. However, following infection by live virus of cells able to support virus multiplication, interferon is usually detectable only after some delay. When chick chorioallantoic membranes were infected with a large dose of influenza

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virus, multiplication of virus occurred within the first 24 hours but no interferon was detected, During the next 24 hours virus multiplication slowed down and interferon was then produced (Burke and Isaacs, 1958b). Wagner (1980) has shown growth curves of influenza virus cultivated in the chick embryo in which interferon appeared in the allantoic fluid 24 hours after the production of viral hemagglutinin. Production of interferon occurring 24-48 hours after virus production was also observed for infection of the mouse brain with Onyong-nyong virus (Hitchcock and Porterfield, 1981) and infection of chick cells with tick-borne encephalitis virus (VilEek, 1961). This delay following infection with multiplying viruses contrasts with the early production of interferon when inactivated or nomultiplying virus is used. One possibility is that the delay allows time for virus inactivation to occur and that the inactivated virus then sets off the production of interferon. Alternatively, it is possible that an individual cell actively supporting virus multiplication produces no interferon until a late stage of virus multiplication is reached, when interferon may then accumulate and help to bring virus production to a halt. 111. PROPERTIES

A. Phystcochemfcal Properties Interferon is nondialyzable and not sedimented on centrifugation at 100,OOO g for 4 hours (Isaacs et al., 1957). Estimates of its molecular weight have been based on its rate of diffusion and its behavior on centrifugation. Porterfield et al. (1960) measured the size of the zones of protection produced in virus-infected chick cells to which were applied at various intervals of time after infection beads containing either chick interferon or viral antibody. The diffusion coefficient of interferon was found to be much higher than that of rabbit antibody and the molecular weight was estimated as less than 80,OOO. Burke (1981) studied the behavior of purified chick interferon in the analytic ultracentrifuge. The interferon behaved in the ultracentrifuge as a single component with a molecular weight of 83,000 and a sedimentation constant of 4.77 S. Little work has been reported on the molecular weight of interferons of other animal species, although they resemble chick interferon in being nondialyzable and not sedimented at 100,OOO g for periods of 1-2 hours. Note added in proof: Recently, new information has appeared on the molecular weight of interferon. Lampson et d.(1963) studied a highly purified preparation of chick interferon and estimated, by means of

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high-speed centrifugation, that it had a molecular weight of 20,00034,OOO. Rotem and Charlwood (1963) carried out studies of the molecular weight of chicken, mouse, and monkey interferons by means of sedimentation in sucrose density gradients along with radioactivelabeled markers of known molecular weight. By use of t h i s technique, all three interferons were found each to have a molecular weight close to that of lysozyme with limits of 13,000-25,000. It seems likely from these findings that the preparation studied by Burke cannot have been purified sufficiently. The protein, glycoprotein, or polypeptide nature of interferon is inferred primarily from the fact that its antiviral activity is greatly reduced or abolished by treatment with proteolytic enzymes, e.g., trypsin (Lindenmann et al., 1957), pepsin (Burke and Isaacs, 1958a), or chymotrypsin (Wagner, 1960).On the other hand, it was not affected by treatment with ribonuclease, deoxyribonuclease, or neuraminidase. Some of its other physicochemical properties are those that might be expected of a protein. According to Lampson d al. ( 1963), one unit of interferon activity in an assay in chick cells was 0.0042 pg. of protein. Interferon is stable on storage at 2O, -loo, or -7OOC. However, the reports of the stability of interferon on heating have been very conflicting. Chick interferon was inactivated on boiling for 5 minutes. In an early report it was found to be inactivated on heating at 6OOC. for 1 hour (Isaacs et al., 1957) but this result may have been due to the pH not having been controlled. On heating at pH 7.2 to 7.4 it resisted heating at 60°C. for 1 hour (Isaacs, 1960b). Wagner (1960j found interferon prepared from chick allantoic fluid to resist heating at 70% for 1 hour, and it is possible that other proteins present in the allantoic fluid may stabilize the interferon to heat. Human interferon was found to have its activity reduced but not abolished by heating at 56OC. for 30 minutes (Ho and Enders, 1959a) and to be completely inactivated by heating for 1 hour at 60°C. at pH 7.8 (Gresser, 1961a), a finding which corresponds to our experience with human interferon. On the other hand, Chany (1981) found human interferon to be completely inactivated at 56OC. for 30 minutes, whereas Mayer (1962) found it to be stable on heating at 60°C. for 1-2 hours. Rabbit interferon was found to resist heating at 56OC. for 30 minutes but to lose activity on heating at 85OC. (Nagano and Kojima, 1958). Mouse interferon was found to be more heat-labile than chick interferon, being inactivated by 60°C. for 1 hour (Henle et al., 1959; Isaacs and Hitchcock, 1960), whereas Glasgow and Habel (1962) reported mouse interferon to be stable after heating at 60°C. for 1 hour. In view of the biological differences in interferons from different animal species discussed below,

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it would not be surprising if they differed in heat stability too, as occurs, for example, in the case of ribonucleases from different sources. HOWever, some of the conflicting reports raise the question of the influence of other constituents present along with the test materials on the apparent heat stability of interferon. Interferon is stable over a wide pH range, from pH 1-10 (Lindenmann et al., 1957). It is also very stable on irradiation with UV light (Burke and Isaacs, 1958a; Nagano and Kojima, 1958; Zemla and VilEek, 1961b).* It can be precipitated by saturated ammonium sulfate (Lindenmann et d.,1957) or by acetone or ethanol (Zemla and VilEek, 1961a,b). Its reported behavior with ether seems to be variable. Most of the reported investigations have been concerned with chick interferon. More investigation is required to know whether interferons from other animal species have similar physicochemical properties.

B. Biological Properties

I. Antigenicity Interferon is antigenically quite distinct from the virus that induced its production (Isaacs et al., 1957). This is such a fundamental point of distinction that it has been included in the definition given at the beginning of this chapter. Interferon appears to be a poor antigen. When inoculated into rabbits or hens either alone or with oil adjuvants or after precipitation with alum, chick interferon did not induce the production of neutralizing antibody (Burke and Isaacs, 1960; Lindenmann, 1960) nor of precipitating antibody ( Belton, personal communication, 1960). Nagano and Kojima (1960) found that a series of injections of rabbit interferon into hens, guinea pigs, and two groups of rabbits produced no neutralizing antibodies; however, a third group of rabbits developed neutralizing antibodies as measured in the rabbit skin. Later Nagano and Kojima (1961) confirmed this finding and also found neutralizing substances in the serum of immunized fowls. Recently Paucker and Cantell (1962) have found that after prolonged immunization of guinea pigs with mouse interferon a very low-titered antibody was found. Antibody could be demonstrated only by using very dilute preparations of interferon. As far as the evidence goes, therefore, interferon appears to be a very weak antigen. The fact that interferon is quite distinct from virus serologically allows the use of viral antibody to inactivate virus without affecting

* Lampson et al. (1963)do not find this to be true of highly purified chick inter-

feron.

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interferon in materials containing both constituents. Other methods that have been used for the same purpose are high-speed centrifugation, treatment at low pH ( 1-2), heat, and UV irradiation. With each virus that is tested it is necessary to be sure that the method of inactivation employed is effective in removing all traces of infectivity, e.g., treatment for 24 hours at pH 2 is effective with most myxoviruses but not with poliovirus. 2. Species Specificity

The first observation of species specificity of interferon was that of Tyrrell (1959), who found that calf and chicken interferons were much more active when tested in cells of the homologous than the heterologous animal species. Subsequently, species specificity has been found between interferons in chick and rabbit cells (Isaacs and Westwood, 1959a), chick and human cells (Ho and Enders, 1959b), and even chick and duck cells (Wagner, 1961). The species specificity is not absolute, the general finding being that interferon is less effective when tested in heterologous cells. However, even this depends on the technique of assay employed; an insensitive assay made it appear that monkey interferon was much more active when tested against vaccinia virus in human thyroid than in homologous cells (Isaacs et al., 1961b). As described in Section II,B, many lines of tumor cells also produce interferons that are more easily assayed in normal cells, even of heterologous species (Chany, 1961), than in homologous cells. Sutton and Tyrrell (1961) have tested interferons of a number of animal species in cells of homologous and heterologous species. Rhesus monkey interferon showed some antiviral activity when tested in calf, human, rhesus, or cynomolgous monkey cells. Human interferon, on the other hand, showed a greater species specificity. Monkey kidney interferon was found to be active when tested in monkey or calf cells, but calf kidney interferon was active in calf but not in monkey cells. Curiously, Sellers and Fitzpatrick (1962) found just the reverse oneway relationship between calf and monkey interferon. This raises the question of whether the species specificity of interferon is an absolute or a variable factor, which may depend on the preparation used, its degree of purification, or other unknown factors. An absence of crossprotection between chick and mammalian interferons was reported by Pollikoff d al. (1962). It is not known whether the species specificity depends on the uptake of interferon by cells or on its behavior at an intracellular site. It is well known that in vivo homologous antibodies are better taken up by cells than antibodies from foreign animal species but it is not yet known

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whether interferon behaves in a similar way. Recently, Gifford (1963a) has found some preliminary evidence that would favor this interpretation. 3. Adsorption

In studies carried out in a tube assay, adsorption of chick interferon to cells was found to be slow (Lindenmann d al., 1957) and similar observations were made by Sellers and Fitzpatrick (1962) for monkey interferon in a test-tube assay, However, Wagner (1961) found much more efficient adsorption of chick interferon when it was applied in very small volumes to monolayers of chick embryo cells. When 0.1 ml. volumes were applied to sheets containing roughly 2 x lo7 cells, 75% of the interferon activity was removed in 20 minutes, Thereafter adsorption slowed and was not complete by 4 hours. The different results found in these methods are probably due to more efficient absorption occurring from a small volume of fluid.

IV. PURIFICATION Although a number of investigations have been carried out in different laboratories on various steps in the purification of interferon, there was, until recently, only one published report on the purification of interferon. This is a report by Burke (1961) describing stages in the purification of chick interferon and some properties of the purified material. The starting material was crude chick interferon containing 150200 pg. of protein/ml., prepared by incubating chick chorioallantoic membranes with UV-inactivated influenza virus in a buffered salt solution. This was first concentrated by precipitation with ammonium sulfate followed by pressure dialysis. Dialysis against pH 2 buffer served as a convenient sterilizing step and also caused precipitation of some heavily pigmented material without loss of interferon activity. On testing a number of different procedures it was found that after chromatography on diethylaminoethyl (DEAE ) cellulose columns at pH 6.6 the eluate could be dialyzed to pH 4.5 and run on to a previously equilibrated column. Interferon was not retained by this column and the biological activity was recovered quantitatively in the eluate. Three other components which had been shown to be present by starch-gel electrophoresis were now found to be retained by this column. The biologically active eluate was next chromatographed at pH 5.8 when a single symmetrical peak was obtained which gave a single band on starch-gel electrophoresis at both pH 8.9 and pH 2.0. Examination in the ultracentrifuge also gave a single component of molecular weight 63,OOO.

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In this work, as well as in the study of certain physicochemical properties mentioned in Section 111, it is not yet known how pure a given preparation of interferon is, nor whether a particular property under investigation might vary as a result of association of interferon molecules with other substances present. With this reservation, the preparation studied by Burke (1961) was found to be a protein containing no nucleic acid and only small amounts of carbohydrate, i.e., 1.6%;hexosamine 2.4%.Since it was retained by DEAE-cellulose columns at pH 5.0 but not 4.5 its isoelectric point was between pH 4.5 and 5.0. The degree of purification achieved was about eO-fold, materials containing protein at a concentration of 6 pg./ml. showing good antiviral activity. However, the total recovery of interferon was low. Hence it is likely that the figure of 20-fold purification achieved may be an underestimate, since it was not possible to be sure how much inactivation of the biological activity of interferon occurred during purification. At the moment there is no way of knowing whether low recoveries of the biological activity of interferon during purification are due to inactivation or to loss of material by coprecipitation with other substances present. Note added in proof: Recently, Lampson et al. (19f33) have published a detailed account of the purification of chick interferon obtained from embryonated eggs infected with influenza A virus. The technique involved ( I ) precipitation with perchloric acid to remove virus and extraneous proteins, ( 2 ) concentration and purification by precipitation with zinc, (3) column chromatography on carboxymethylcellulose, followed by ( 4 ) zone ionophoresis on pevikon. One unit of interferon activity corresponded to 0.0042 pg. of protein, a considerably higher degree of purification than had been achieved hitherto. V. MODEOF ACTION A. Site of Action in Virus Growth Cycle

At an early stage of the work it was clear that interferon acted by rendering cells resistant to virus multiplication and did not act on extracellular virus. This could be shown by the fact that in an assay in chick membrane fragments interferon exerted its full effect only when it was incubated with cells for some hours before infecting with challenge virus (Lindenmann et al., 1957). Absence of direct interaction of interferon and virus was observed by Ho and Enders (1959a,b) and Vilbk (1960).

16

ALICK ISAACS

In support of this conclusion are the findings of Isaacs and Burke (1958) that cells treated with sufficient interferon to induce 95%inhibition of virus growth were able to take up either live or inactivated virus which gave rise to good yields of interferon. Further support comes from the findings of Wagner (1960, 1961) that, when studied in a single cycle of v i r u s growth, interferon showed an antiviral action even when applied to cells 2 hours after infection had been initiated. Wagner’s results also showed that interferon does not act by affecting adsorption or uptake of virus. Experiments which revealed no effect of interferon on virus adsorption when tested directly are described by Wagner (1960, 1961) and by Isaacs (1960a). The studies of Grossberg and Holland (1962) on poliovirus demonstrate that interferon does not act by inhibiting release of virus from cells. It seems clear, therefore, that interferon inhibits virus replication at an intracellular sitk. Further investigation suggested that interferon acts ,at an early stage of virus growth. Thus it inhibited not only the prodpction of mature virus particles but it also inhibited to a correspondin4 degree the synthesis of cell-associated virus antigens such as the yaccinial hemagglutinin (Isaacs et aZ., 1958), the influenza1 nucleoprotein soluble antigen, and the viral hemagglutinin (Burke and Isaacs, 1960). More precise evidence comes from observations that show that interferon inhibits the synthesis of viral RNA. De Somer et qZ. (1962) found that in chick cells treated with interferon and infected with Western equine encephalitis virus there was inhibition of the synthesis of viral RNA and of mature virus. These results were confirmed by Ho (1962c), who noted, in addition, that the synthesis of RNA was inhibited slightly less than the synthesis of mature virus particles. This last finding suggested that interferon inhibited indirectly the synthesis of viral RNA, or alternatively, that it inhibited the synthesis of another viral constituent in addition to viral RNA. Inhibition of the replication of infective viral RNA was first observed by Grossberg and Holland (1961, 1962) and has been confirmed by others. In these experiments it is essential that a single cycle of virus growth should be studied. If this precaution is omitted it is possible that a substance under test might not inhibit the replication of infective RNA but inhibit the multiplication of mature virus particles formed after the first cycle of virus growth. In order to be sure that a single cycle of virus growth was observed, multiplication of poliovirus RNA was studied in chick embryo fibroblasts (Grossberg and Holland, 1961; Ho, 1961) or in the chick embryo (De Somer et d.,1962). Interferon was found to inhibit the replication of viral RNA and showed no direct action on the extracellular RNA (De Somer d al., 1962). These findings

INTERFERON

17

suggest that interferon acts after the virus particle has been adsorbed to and penetrated cells and after its protein coat has been removed, but before its viral nucleic acid has been replicated. It is not yet possible to say very much about the site of action of interferon within the cell, although this point is mentioned briefly in the following sections.

B. Conditions Required for Action of Znterferon For the full action of interferon, some hours of incubation at temperatures around 37OC. are required. This was shown by experiments in which cells were allowed to adsorb interferon for 3 hours at 37OC. when they were washed and then incubated for 21 hours at either 2O or 37OC. before virus challenge. Less viral inhibition was found in the cells kept at 2OC., suggesting that a metabolic process in the cells requiring some hours’ incubation at 37OC. was required before the action of interferon was fully established (Lindenmann et d.,1957). An essentially similar result was found by VilEek and Rada (1962). These findings are not easy to reconcile with the observations of Wagner that interferon shows some antiviral action even when given 2 hours after the initiation of virus infection. It may be that when large doses of interferon are used only a short period of time is required to observe some antiviral action, but that a longer time is required when small amounts of interferon are assayed. The duration of action of interferon seems to be very variable and to depend greatly on the metabolic state of the cells. Isaacs and Westwood (1959b) studied its duration of action by exposing chick cells suspended in a maintenance medium to interferon on a single occasion, infecting them with West Nile encephalitis virus, and observing the yield of virus at daily intervals. Under these conditions the cells showed no cytopathic effect and released no virus during a period of 11 days. However, when the medium was enriched with calf serum and chick embryo extract the cells degenerated rapidly and released virus into the medium. The explanation that was suggested was that cells kept in a maintenance medium were unable to divide and retained sufficient interferon to inhibit virus growth. However, on addition of nutrients cell division commenced and the intracellular concentration of interferon fell below that required to inhibit virus replication. An alternative explanation would be that interferon has a greater antiviral action in resting cells than in metabolically active cells. The cells used are a second factor and Sutton and Tyrrell (1961) found that calf kidney and rhesus monkey kidney cells treated with interferon on a single occasion showed some resistance to virus infection

18

ALICK ISMCS

for 5 days. Wagner (1981)found that chick cells treated with interferon developed resistance to infection 4 to 5 times more quickly than they regained susceptibility on further incubation in interferon-free medium. A rapid regaining of susceptibility in chick embryo cells treated with interferon was reported by Bader (1962); however, the particular assay used measured susceptibility to Rous sarcoma virus, an assay which takes roughly 7 days to read, so that Bader’s results do not s&m to be in disagreement with those of other workers. It is implicit in these conclusions that interferon does not replicate and this has been demonstrated experimentally. At the same time it was observed that no interferon could be recovered by disrupting cells that have taken up relatively large amounts (Isaacs et al., 1957; Wagner, 1W)The . significance of this finding is not at present clear. C . Mechanism of Action Chick cells treated with interferon and then infected with either live or irradiated influenza virus were found to give good yields of interferon although their ability to support virus multiplication was greatly inhibited (Isaacs and Burke, 1958). This suggested that the action of interferon might be described as a redirection of the pathway of infecting virus from the synthesis of virus toward the synthesis of interferon. VilEek and Rada (1962)found that chick cells treated with interferon and infected with tick-borne encephalitis virus showed inhibition of the production of virus and interferon, but Ruiz-Gomez et al. ( 1963), in studies with Chikungunya virus in chick cells, found essentially similar results to those of Isaacs and Burke. The reason for these variable results is not yet known. It may depend on whether, under the experimental conditions studied, some virus multiplication is necessary before interferon is produced. Alternatively the results may reflect whether the interferon preparation used contains some inactivated virus, as suggested by Ho and Breinig (19s2). This would seem to be an unlikely explanation for influenza virus inactivated by pH 2 treatment, since this has the effect of abolishing the hemagglutinating activity of the virus, and presumably, therefore, its ability to adsorb to cells. The suggestion that interferon treatment of cells redirects the pathway of the infecting virus from the synthesis of new virus toward the synthesis of interferon may be an expression in biological terms of the biochemical findings, described below, that cells treated with interferon are able to grow and divide and can presumably synthesize normal cellular proteins and nucleic acids at a normal rate, and yet are unable to support the replication of viral nucleic acid.

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19

Numerous hypotheses have been put forward to account for the mechanism of action of interferon. Wagner (1960) thought that interferon behaved like a basic protein which could combine with viral nucleic acid once it was released from viral protein in the cell. The findings of Burke (1961) on its isoelectric point and the evidence of De Somer (1962) that interferon does not inactivate viral nucleic acid when mixed with it directly would not favor this hypothesis, although De Somer suggests that interferon may stimulate cells to produce a basic protein. The author has been guilty of propounding from time to time hypotheses among which is the suggestion that interferon inhibits an oxidative process that supplies energy for virus synthesis. This hypothesis was first suggested by the observation that chick embryo cells treated with large doses of interferon showed a slight increase in oxygen uptake and a greatly increased glycolysis (Isaacs, 196Ob). It was di5cult to be sure that it was the interferon which was responsible, but in favor of this conclusion a "mock" preparation prepared in the same way, but omitting the virus, showed no such activity and the active factor shared with interferon a similar heat stability, stability on treatment at pH 2, and absence of dialyzability. Stimulation of glycolysis in chronically infected cultures shown to be producing interferon was observed by Green et al. (1958) and has since been observed by many workers. Increased glycolysis was reported by Allison (1961) in mouse cells treated with mouse interferon and by Gresser (1961~) in human cells treated with human interferon. Levy et d. (1962) commented on repeated observation of increased glycolysis produced by chick and mouse interferons but found that this effect lacked the species specificity found in the antiviral actions of these preparations. Zemla and Schramek (1962a) found increased glycolysis produced both by a preparation of interferon and by a mock interferon preparation lacking antiviral activity and concluded that it was not the interferon that was stimulating glycolysis. This is not a necessary conclusion since it is possible that a number of different substances might stimulate glycolysis. However, at the moment it is not clear whether the stimulation of glycolysis observed by a number of workers is due to interferon, to some closely related substance, or to unrelated substances (cf. Lampson et aZ., 1963). The same reservation is required with regard to other metabolic changes observed in cells treated with interferon. It will not be possible to decide on the significance of these findings until they can be repeated with highly purified preparations. Substances that uncouple oxidative phosphorylation stimulate cells to increased glycolysis while at the same time the oxygen uptake may be

increased. The observation that dinitrophenol, an uncoupler of oxidative phosphorylation, did not inhibit the growth of poliovirus in HeLa cells when given in doses that inhibited virus multiplication in normal cells (Gifford and Blakey, 1959) was made at about the same time as Ho and Enders (195913) found that interferon was produced in HeLa cells but had to be assayed in normal human cells since HeLa cells were relatively insensitive to its antiviral action. These resemblances between the actions of interferon and agents that uncouple oxidative phosphorylation were added to when it was found that a virus that was particularly sensitive to interferon was also more sensitive to the action of four different uncoupling actions, whereas a virus more resistant to interferon was also more resistant to the uncoupling agents (Isaacs et al., 1961a). This is indirect evidence that interferon may act by uncoupling oxidation from phosphorylation, but at the moment there is no direct evidence on this point; if such a mechanism were operating it would be necessarily at a localized site within the cell, possibly at a nuclear site. The significance of the evidence of Zemla and Schramek (1982b) that interferon inhibits virus growth in chick cells under anaerobic conditions depends on how sure one can be that complete anaerobiosis was obtained. Most workers have found that virus growth in normal cells is poor under low oxygen tensions and Baron et al. (1961) observed that different viruses have different oxygen requirements, as judged by the depth to which virus will grow in chick cells kept in a culture tube filled with agar. Those viruses with the highest oxygen requirements were the most sensitive to the antiviral action of interferon and the converse was equally true (Isaacs et al., 1961b). In addition it was found that increased oxygenation of cultures tended to diminish the antiviral action of interferon, whereas reduced oxygenation had the reverse effect. This might suggest that interferon was inhibiting an oxidative process that supplied energy for viral synthesis; apparently viruses differ in their oxygen requirements, those with the highest oxygen requirements being most readily inhibited by interferon. Also in favor of this interpretation is the finding that tumor cells and the cells of young embryos (see Section I1,B) are less sensitive to the antiviral action of interferon than normal cells. Tumor cells and the cells of young embryos are generally less dependent on oxidative processes as a source of energy than normal cells. Mosley and Enders ( 1962) found that polioviruses grown in monkey kidney cells had a bicarbonate requirement which vaned with the virus strain. In general, avirulent strains multiplied very poorly when grown in tubes plugged with cotton wool, which allowed accumulated carbon

INTFZFERON

21

dioxide to escape, whereas the growth of virulent viruses was little affected. Evidence was produced that the decreased rate of growth in cotton-plugged tubes corresponded to the low plating efficiency found by Vogt et d. (1957) with avirulent polioviruses grown with an agar overlay containing a low bicarbonate concentration (the d marker). The avirulent and virulent viruses used correspond fairly well, as discussed in Section VI, with strains showing greater or lesser sensitivity to the antiviral action of interferon. It appears, therefore, that certain virus strains that are sensitive to the antiviral action of interferon have relatively high oxygen and high bicarbonate requirements compared with strains that are less sensitive to interferon. Gifford (1963b) has studied the oxygen and bicarbonate requirements of certain viruses and has found that both increased oxygen and bicarbonate have the effect of increasing the depth to which a virus grows in a tube culture filled with agar. The effects of oxygen and bicarbonate were found to be additive, so that increased bicarbonate could not replace oxygen. It is possible that an oxidative process required for virus synthesis may be dependent not only on oxygen but also on bicarbonate or carbon dioxide. If interferon were blocking the supply of energy needed for viral synthesis it might be anticipated that with small doses of interferon, once the antiviral effect had worn off, the viral RNA would be able to resume its replication, so that delayed virus growth would be observed. This has in fact been found by Mayer et al. (1961, 1962), De Somer et al. ( 1962), and Grossberg and Holland ( 1962). A similar observation has been made by E. Heller (personal communication, 1962) with regard to polyoma virus. With larger doses of interferon virus growth is suppressed and in vaccinia1 infections of the rabbit skin, for example, interferon does not merely delay but prevents the appearance of viral lesions ( Isaacs and Westwood, 1959a). Whether cellular nucleases or other mechanisms are responsible for the suppression of infection is not known, Also, in agreement with the suggestion that interferon inhibits the supply of energy, is the fact that it does not block the uptake of virus and the release of nucleic acid from protein but that it inhibits the replication of viral nucleic acid. This would be one of the &st steps requiring an energy supply from the host cells. Unfortunately, it is not yet possible to provide more than indirect evidence that would support this or any other hypothesis on the mode of action of interferon.

D. EflBcts of Interferon on Cells Three types of effects of interferon on cells have been studied: morphological changes, alterations in the growth rate of cells, and biochemical changes.

22

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1. Morphological Changes With most tissue culture systems an absence of any significant morphological change has been noted in cells treated with interferon. Wagner and Levy (1960)studied chick embryo fibroblasts protected by interferon and infected with Eastern equine encephalitis virus. Cells stained with acridine orange showed normal architecture and a distribution of DNA and RNA that was indistinguishable from that of normal cells. Despite the viral infection mitotic figures were readily seen in cells pretreated with interferon, The picture was in striking contrast to the rapid degeneration found in cells that were infected with virus but not treated with interferon. One interesting morphological change in cultures of human amnion cells treated with interferon was described by Gresser (1961~).Two to three days after the introduction of interferon many of the normally polygonal cells became fusiform and resembled whorls of fibroblasts, so that recognition of the original cell type was difficult. The changes were readily reversible, the cells resuming their normal appearance within 24 hours of removing the interferon from the medium. Only preparations with antiviral activity showed this effect and the active factor resembled interferon in many of its physicochemical characters. Treatment of primary cultures of human kidney cells or of a continuous cell line derived from human amnion cells produced no morphological changes. 2. Growth Rate of Cells Treated with Znterfmon Baron and Isaacs (1962) found that cultures of primary human thyroid cells treated with about one hundred 504; inhibitory doses of interferon were resistant to the multiplication of vaccinia virus but were nevertheless able to grow and divide normally. The interferon was present from 24 hours after the cells were first seeded in tubes and the cells formed a complete sheet at about the same time as the untreated control cells. Paucker et al. (1962) made a careful study of the growth rate of L cells kept in suspension for periods of 25 days and treated with different amounts of interferon. Cells treated with 10 units of interferon showed a very slight inhibition of growth rate. Cells treated with 100 units showed slight inhibition for the first 8 days with steadily increasing inhibition thereafter. Cells treated by continuous exposure to 700 units showed almost total cessation of cell growth. When the interferon was removed, even after prolonged contact with L cells, there was a gradual recovery with resumption of the normal rate of growth. Treatment of cells by a single exposure to 2000 units followed by removal of the interferon led to a short-lived depression of cellular

INTERFERON

23

multiplication which lasted for about 3 days after which the normal rate of growth was resumed. 3. Metabolic Changes in Cells Treated with Interferon

The increased glycolysis and uptake of oxygen in cells treated with interferon were referred to in Section V,C. Few other changes were described and Levy et al. (1962) reported a number of negative findings in their attempts to find biochemical changes in cells treated with interferon. Recently, however, Levy and Baron (1963) have made some interesting new observations. Exposure of chick embryo fibroblasts to actinomycin D inhibits RNA metabolism by about 90-95%, the metabolism being measured by uptake of H3-uridine into phenol-released RNA. Treatment with interferon blocked about 50-75%of the remaining RNA metabolism. If actinomycinresistant RNA metabolism is non-DNA-dependent then interferon appears to inhibit that small fraction of RNA metabolism in normal cells. What function this RNA has in normal cells is not yet known. In cells infected with Sindbis virus, at 34 and 4$ hours after infection, in the presence of actinomycin, virus-infected cultures took up much more H3-uridine into RNA than did uninfected cultures. Presumably much of this RNA synthesis can be attributed to the formation of viral RNA and it was completely inhibited by inclusion of interferon in the medium. These results suggest that a non-DNA-dependent RNA synthesis is the target for the action of interferon. It is possible that interferon acts either on the supply of energy for this particular RNA synthesis or that it inhibits the formation or action of a polymerase or other enzyme required for the formation of this RNA.

E . Production and Action of Interferon at the Cellzrlar Level An early observation was that the degree of virus inhibition produced by interferon was, within certain limits, independent of the dose of challenge virus, and depended only on the amount of interferon used (Lindenmann et al., 1957; Lindenmann and GBord, 1963). Thus, a given dose of interferon produced the same degree of viral inhibition when the dose of challenge virus was vaned over a 100-fold range. This result resembles that found by Fazekas de St.Groth and Edney (1952) for viral interference produced by heated influenza virus. Ho (1962a) has found that when chick cells were treated with a small dose of interferon and then infected with vesicular stomatitis virus and the values for multiplicity of virus input plotted against the number of cells required to produce one infectious center the proportion of protected

24

ALICK ISAACS

cells was greater at low virus inputs. This suggests that the protective effect of interferon in terms of prevention of cell infection may be overcome by large virus inocula. These results offer a possible explanation of some factors in the development of a vinis plaque in cells treated with interferon. When a low virus multiplicity is used the first cell infected will have received a single virus particle. However, when this cell produces large numbers of virus particles which infect its neighbors those secondarily infected cells will receive a much higher dosage of virus. This may help to explain why many plaques found in cell sheets treated with a low dose of interferon show a relatively normal plaque size and appearance. Bellett and Cooper (1959) studied an interfering component, probably interferon, that was produced by chick cells infected with vesicular stomatitis virus. When the concentration of interferon was plotted against the logarithm of virus yield relative to the maximal yield, a negative exponential relationship was obtained between dose of interferon and cells remaining uninterfered. This result was found with relatively low virus doses, i.e., multiplicity of 2.5, and it suggests that one particle” of interferon per cell was sufficient to induce cell protection. However, this one-hit curve does not tell us how many molecules of interferon per cell must be present before one effective particle will be found. Cooper and Bellett (1959) found that the total virus yield from interferon-treated cultures was reduced by about the same factor as the number of cells able to release virus. This implies an all-or-none response of cells which would either produce a normal yield of virus or show an absence of virus production. These two conclusions have been confirmed by some workers and disputed by others. Bader (1962) measured the yield of Rous virus from chick embryo cells treated with different dilutions of interferon and concluded that one unit of interferon was sufficient to inhibit replication of a single particle of Rous virus. Ho (1962a) studied the number of plaques produced by vesicular stomatitis virus in cells treated with different dilutions of interferon. He did not find a linear response except possibly at low doses of interferon. At high doses of interferon there was a relatively decreased inhibitory effect. Lindenmann and Giflord (1963) plotted the dose-response curve of the logarithm of interferon concentration against plaque count with vaccinia virus, They found an S-shaped curve which was linear over a range of interferon concentrations, but which became flatter at high concentrations of interferon. Possibly the result of experiments of this kind may depend on whether an assay measuring virus yield or an assay measuring plaque production is used.

INTERFERON

25

Results similar to those of Cooper and Bellett (1959) on the all-ornone response of cells to interferon were described by Ho (1961) for inhibition of the development of poliovirus RNA in chick embryo fibroblasts. Later Ho (1962a), in studies with chick cells infected with vesicular stomatitis virus, observed that the reduction in total virus yield was greater than the reduction in the number of infective centers. He also repeated his earlier experiments with poliovirus RNA and now found that the ratio of virus yield to infective centers was usually lower in interferon-treated than in control cultures. These findings suggested that in addition to suppressing virus development in cells, interferon can also lead to a reduced output of virus in certain cells. A similar conclusion was reached by Wagner ( 1961), who observed a prolonged latent period in cells treated with interferon. Wagner suggested that if a reduced virus yield were to be attributed to a normal yield of virus from a minority of the cells the release of virus should have occurred at the same time as in the control cells. However, a delay in the appearance of virus in cells treated with interferon was discussed in Section V,C, and this was advanced to support the view that interferon acts by inhibiting the synthesis of viral RNA, which can be resumed in certain cases once the effect of interferon has passed off. This delay is stressed in the study by Gifford et al. (1983) of the times at which plaques appear in cells treated with interferon. Thus, the apparently greater reduction in the yield of virus in proportion to the reduction in the number of cell yielders described by Ho (1962a) may simply be an expression of a delay in virus synthesis in a minority of cells and a complete suppression of virus synthesis in the majority. Investigation of samples taken at Werent times could settle t h i s question. Cantell et al. (1962) found that L cells treated with interferon and infected with vesicular stomatitis virus frequently showed the development of viral antigen within the cells in the absence of production of infective virus. However, Cantell et al. point out that vesicular stomatitis virus has a toxic property for these cells and that it would be unwise to generalize from the results with t h i s virus. There is evidence from the work of Gresser (1961a) and Gresser and Enders (1962) that interferon which protected against virus multiplication gave only very weak protection of human amnion cells against the toxic effect, which is not associated with virus multiplication, and is produced by Sendai or Sindbis viruses. This is a very similar finding to that of Cantell et d. (1962) and it recalls an earlier observation of Isaacs and Fulton (1953) that viral interference induced by irradiated influenza virus was much more effective in the allantoic than in the chorionic cells of the chick chorioallantoic membrane. In the allantoic cells a complete cycle of

26

ALlCK ISAACS

influenza virus growth was found and this was readily inhibited, whereas in the chorionic cells only an incomplete cycle of virus growth occurs and this is much more resistant to viral interference. It seems important in studies of the effects of interferon at the cellular level to distinguish complete cycles of virus growth from incomplete cycles. The results of a virus-cell interaction in a cell that has taken up interferon will presumably depend on the amount of interferon taken up, the stage in the virus cycle at which it is able to exert its action, whether it has had sufficient time to exert its full antiviral action, or whether its action has been reversed. It will depend, too, on the multiplicity of the infecting virus and possibly on the metabolic state or the stage of division of the cell. More precise information will require studies of individual cells.

VI. INTERFFBON AND RECOVERYFROM Vmus INFECTION Recovery from virus infection has been investigated in uitro, in the chick embryo, and in mature animals. In the first two cases, cellular factors in the recovery process can be studied free from any complications of antibody production or delayed hypersensitivity, but study in mature animals is a more complex problem, The factors involved in this last case are considered in more detail in the review by Baron (this volume).

A. Recovery from Virus Infection in Vitro Bang and Gey (1952) studied the growth of Eastern equine encephalomyelitis virus in 13 established strains of rat cells. There was great variation in susceptibility with extremes formed by a normal cell strain and its specific malignant cell derivative. The normal cells were resistant to virus growth, whereas the malignant cells were rapidly destroyed with the production of large yields of virus. Between these two extremes cell strains continued to support virus growth for periods of several months. Destruction of cells in these cultures seemed to be focal in nature and Bang and Gey (1952) suggested that inhibiting factors might be present in the media used. One possible explanation for cellular resistance found in chronically infected cultures is that virus acts selectively, destroying the most susceptible cells and favoring the growth of resistant cells, Bang et al. (1957) found that chronically infected cultures could be cured of virus infection by raising the temperature to 37OC., whereas lowering the temperature to 31OC. favored maintenance of the chronic infection. However, cultures that showed recovery from viral infection were consistently susceptible to 3 successive reinfections, implying that the rela-

INTERFERON

27

tive growth of resistant cells was not an explanation for the recovery in this case. An alternative suggestion, discussed below, that interferon might be playing a role in the behavior of these cultures, is in line with Bang and Gey’s finding that tumor cells were more susceptible than normal cells, since tumor cells are usually less sensitive to the antiviral action of interferon than normal cells (see Section I1,B). Also, the poor growth of virus at higher temperatures would fit with the observation, discussed further below, that a rise in temperature may favor the production of interferon. Ho and Enders (1959a,b), Henle et a2. (lS59), and Mayer (1962) studied chronic virus infections in vitro and observed the accumulation of interferon in the media of these cultures. This raised the possibility that interferon might be responsible for maintaining the state of chronic infection. This question was posed by Glasgow and Habel (1962) in a series of elegant experiments. They studied a mouse cell line that was chronically infected with vaccinia virus and showed that it was possible to stabilize the chronic infection, or to produce either complete cure of the infection or total cell destruction. In order to make the infection take one or other course all that was required was to raise or lower the concentration of interferon in the medium, or keep it relatively constant, by changing the medium at different intervals of time. Treatment of the culture with small doses of trypsin, which inactivates interferon, had the same effect as frequent changing of the medium. Chany (1961) had also found that in chronic infection of KB cells with parainfluenza 3 virus treating the cells with trypsin led to rapid cell destruction. In passing, there would seem to be some contradiction in the suggestion that chronic infections of certain tumor cells could be mediated by interferon, since many tumor cell lines are relatively insensitive to the antiviral action of interferon added to the medium. However, the low sensitivity of tumor cells to exogenous interferon might be compatible with a greater sensitivity to endogenous interferon produced at a site close to its site of action. An interesting observation from studies of cultures chronically infected with a myxovirus was the finding that less than 10% of the cells contained any evidence of the presence of virus, yet the whole culture was resistant to challenge with vesicular stomatitis virus (Henle et al., 1959). The significance of these results is emphasized by the finding of Gresser and Enders (1962) that when two different kinds of human amnion cells were mixed, one of these being sensitive to the destructive action of Sindbis virus and the other being resistant, the mixed culture showed resistance to virus infection. Even the presence of a minority of resistant cells was able to protect the sensitive majority of cells. It was

28

ALICK ISAACS

found that on infection of the resistant cells large amounts of interferon were produced which could then protect the susceptible cells. It seems clear from this evidence that interferon plays an important role in a number of examples of cellular resistance to virus infection in vitro, as shown by recovery from infection or the maintenance of a chronic state of virus infection. We can return here to the question mentioned in Section I1,D-in a culture infected with ‘live” virus and producing both new virus and interferon, is it likely that single cells are producing both virus and interferon at the same time or is it more likely that some cells are producing virus only at a given time, while others are producing interferon only? The mixed cultures of Gresser and Enders (1962) provide a model of a situation in which a population of cells reacts to infection by one virus in two different ways. In this example, production of interferon by a minority of cells has the effect of protecting the whole culture. In the example provided by Henle et aZ. (1959), the minority of cells were producing virus and it is possible that the majority were producing interferon. In most virus infections, it is not known how much variation there is in the response of individual cells. However, study of the cellular changes induced in bovine cell cultures by infection with high multiplicities of influenza virus shows that only a proportion of the cells in infected cultures show pathological changes detectable by staining with acridine orange or fluorescent antibody or the presence of viral antigen as revealed by hemadsorption (Niven et d.,1962). This recalls the finding of Magee and Sagik (1959) that when chick embryo cells were infected with Newcastle disease virus at a multiplicity of 5 or 10 only about 6O!Z of the cells could be shown to yield virus. If it should prove to be correct that in a normal virus infection, cells infected with virus particles that are unable to multiply respond by producing interferon, whereas cells do not produce interferon while they are supporting virus multiplication, such findings would fit well with the observations discussed in Section II,F of the rapid liberation of interferon from cells treated with inactivated virus, in contrast with the delayed production by cells which are actively supporting virus multiplication.

B. Recovery from Virus Infection in the Chick Embryo It has been known for some time that the results of infecting chick embryos with many different viruses depend to a large extent on the age of the embryo at the time of infection (see Beveridge and Burnet, 1946). In general, susceptibility to the lethal action of many different viruses decreases as the embryo ages. When it was found that both sensitivity to the antiviral action of interferon and the ability to produce

INTERFERON

29

p o d yields of interferon on vinis infection increase as the embryo ages (Isaacs and Baron, 1960), an opportunity was presented to see whether resistance to virus infection and development of the interferon mechanism were related. Since the chick embryo does not produce antibody and does not show delayed hypersensitivity, any relationship of interferon to recovery from virus infection could be studied in relative isolation. When this comparison was made it was found that resistance to infection with four different viruses and sensitivity to the antiviral action of interferon in uitro were related to aging of the chick embryo in a very similar manner (Baron and Isaacs, 1961) , Both of these factors were low in embryos of under 7 days and both showed a sharp increase between 7 and 10 days, followed by a much more gradual increase. An increased resistance to the lethal action of mumps virus (Cantell, 1961b) and a considerable reduction in the ability of chick embryos to support the growth of poliovirus RNA (Denys and Prinzie, 1962) were also found to begin at about the seventh or eighth day, These findings favor the conclusion that the interferon mechanism plays an important role in the ability of the developing chick embryo to recover from virus infection. C . Recotiery from Virus Infection in Adult Vertebrates This subject is dealt with in detail in the article by Baron (this volume), who has reviewed the evidence that recovery from virus infection cannot be accounted for solely in terms of production of antibody. In this section a short summary of some points of interest with regard to interferon will be given. Production of interferon in the course of virus infections in tiitio was observed by Nagano and Kojima (1958) in vaccinia1 infection of the rabbit skin, by Isaacs and Hitchcock (1960) and by Link and Raus (1961) in the course of influenza virus infection of the mouse lung, by Hitchcock and Porterfield (1961) during infection of the mouse brain with an arbovirus, and by Friedman et (11. (1962) during vaccinial infection of the skin of the guinea pig. The peak of interferon production was found to occur early in infection, either at the time of the peak of virus production or very shortly afterward, whereas the peak of antibody production was much later. In trying to assess the importance of the production of interferon on the course of a virus infection in duo it was interesting to see what effect inhibiting interferon production would have on the course of the infection. One such inhibitor is increased oxygenation, which was found to inhibit the antiviral action of interferon in uitro (Isaacs et aZ.,

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1961b), a finding which is in line with the suggestion that interferon acts by inhibiting an oxidative process (see Section V,C). Increased oxygenation was found to show an adverse effect on the course of influenza viral infection of mice in uiuo, as shown by a higher mortality and a shortened incubation period of lethal pneumonia (Sawicki et al., 1961). A second inhibitor of interferon production is cortisone, which inhibited both the production and action of interferon in chick cells (Kilbourne et al., 1961) but only its production in rat cells ( D e Maeyer and De Maeyer, 1963). Cortisone has a detrimental effect on the course of many viral infections, but since it may also affect antibody production and delayed hypersensitivity, it is more difficult to assess how much its detrimental action in virus infections is due to its effect on the interferon response. In many virus infections of animals resistance to a number of virus infections increases with age. This was observed to be the case in infection of mice with parainfluenza 1 (Sendai) virus. Sawicki (1961) has shown that in the course of aging mice develop an increased ability to eliminate this virus from their lungs and this is accompanied by increased production of interferon.

D. Virus Virulence Enders (1960) commented on the higher yield of interferon from cells infected with an avirulent strain of measles than from cells infected with a virulent strain. He suggested that this relationship might be a more general one which could yield an interesting clue to the nature of virus virulence. Since this first report, many examples of a similar nature have been observed. De Maeyer and Enders (1963) found that 5 strains of poliovirus of low virulence induced production of interferon, whereas with 4 virulent strains no interferon could be detected. Ruiz-Gomez and Isaacs (1963a) studied production of interferon in chick embryo cells infected with a variety of different viruses. In general it was found that viruses that were most virulent for the chick embryo produced less interferon than viruses of lesser virulence. If cellular susceptibility to virus infection can be thought of as the mirror image of virus virulence it is of interest that Glasgow and Habel (1962) observed that mouse cells that showed lesser susceptibility to vaccinia virus produced more interferon than cells that were more susceptible to the same virus. Again, Ruiz-Gomez and Isaacs (1963b) noted that Newcastle disease virus grew well and produced plaques in chick embryo cells in which, however, very low yields of interferon were found. The same virus grew poorly in human thyroid cells but produced large yields of interferon. Virus virulence

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and interferon production thus share a characteristic in common, in that they do not describe an isolated property of a virus, but only a property of a virus in relation to a particular population of cells. It is rather difficult to compare production of interferon by different viruses since the yield of interferon depends on the dose of virus inoculated, the temperature of incubation, the pH, and other conditions of culture. A measurement which seems to be less affected by these variables is the sensitivity of a virus to the antiviral action of interferon. It has been shown that there is quite a good correspondence between the sensitivity to interferon of a number of viruses grown in chick cells and virulence for the chick embryo, those viruses that are least sensitive to interferon being the most virulent ( Ruiz-Gomez and Isaacs, 1963a). Virulence therefore seems to be related to an ability of a virus either to avoid stimulating the production of interferon or to be relatively insensitive to the action of the interferon produced. One apparent exception found was Kumba virus ( Ruiz-Gomez and Isaacs, 1963a), which produced good yields of interferon and was sensitive to its antiviral action in uitro, yet was highly virulent for the chick embryo. However, recent investigations suggest that the virulence of Kumba virus can be accounted for within the same theoretical framework by postulating that this virus multiplies so rapidly that it is able to outstrip its production of interferon. Indirect evidence favoring this interpretation is provided by the fact that whereas most avirulent viruses, when inoculated on to chick cell monolayers at high virus doses show a prozone, Kumba behaves like a virulent virus, such as Newcastle disease virus, and does not show a prozone. The prozone seems to be due to the fact that with large virus doses, sufficient interferon is produced early, to inhibit the cytolytic action of the virus in these cells. With Kumba virus, although good yields of interferon are produced, this presumably occurs too late to inhibit the cytolytic action of the virus. More convincing evidence of the relationship between virus virulence and the interferon mechanism comes from studies of virus mutants of differing virulence. Wagner (1962) compared the behavior in L cells of mutants of vesicular stomatitis virus of differing virulence for mice. The more virulent virus produced less interferon and was less sensitive to the antiviral action of interferon in uitro than the less virulent virus. A similar result was obtained by Finter (1962) with variants of Semliki Forest virus a t different stages of adaptation to grow in calf kidney cells. A most significant study is that of Thiry (1962) who prepared a number of “ r e d mutants of Newcastle disease virus by treating virus particles with nitrous acid. Red mutants give rise to “plaques” that are intensely colored by neutral red and they show less virulence for chick embryos and mice than the parent virus. Treatment with ethyl ethane

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sulfonate had just the reverse of the effect of the treatment with nitrous acid. These mutants could be arranged in increasing order of virulence as measured by lethality after intracerebral inoculation of mice and inoculation into the allantoic cavity of chick embryos. Thiry found that the lower the virulence the greater the yield of interferon induced, the two properties showing a very close correspondence.* It is obviously not possible to account for virus virulence solely in terms of the interferon mechanism. However, the above results would suggest that in many examples of virus virulence that have been studied, interferon production and action appear to play a significant role. A question of interest that arose is whether a virulent virus is able to avoid stimulating the production of interferon or is able to block actively its production or action. Lindenmann (1960) found that production of interferon by chick chorioallantoic membrane fragments, which is readily induced by infection with heated or UV-irradiated influenza virus, could be blocked by simultaneous infection with live virus. The live virus could be given before, along with, or even 1 hour after the inactivated virus and was still able to induce what Lindenmann called inverse interference. In investigations of inverse interference in chick embryo cells infected with an arbovirus, Ruiz-Gomez et al. (1963) observed that viruses virulent for the chick embryo showed inverse interference, whereas less virulent viruses did not. On the basis of these and other findings the hypothesis was put forward that when a virus particle enters a cell it either stimulates the production of interferon and fails to multiply, or alternatively it inhibits the production of interferon and proceeds to multiply. A number of cultural conditions were mentioned which tended to favor one or other course. Apart from the virus strain and the cells, raising the temperature, lowering the pH or possibly the bicarbonate content (De Maeyer and De Somer, 1962), lowering the oxygen tension, or pretreating the cells with interferon all seeemd to favor the production of interferon relative to the production of virus (Ruiz-Gomez et al., 1963). As discussed below, Heller (1963) has found that minute doses of actinomycin D have just the reverse effect. The interpretation implied in these findings is that one aspect of virus virulence is the ability of a virus to grow despite the normal cellular defense mechanism, i.e., the production of an antiviral substance. Other interpretations of these findings are possible but the interpretation proposed has the advantage of fitting logically with present ideas of virus virulence.

* Sellers (1963) has now found that foot-and-mouth disease vinises of differing virulence also show a corresponding variation in sensitivity to, and production of, interferon.

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VII. THE ROLE OF INTERFERON IN NORMALC E m In trying to speculate on the possible role of interferon in normal cells the assumption has been made that its antiviral action is incidental to another, more basic action. On the basis of this assumption some interesting results have emerged which would tend to support this point of view. However, any understanding of the role of interferon in cells in the absence of virus infection is still at a rudimentary stage. One question which was of interest is what is the stimulus that induces cells to make interferon. Since the production of interferon can be initiated by RNA and DNA viruses, and in the absence of virus multiplication, it seemed that virus protein or virus nucleic acid must be the stimulus (since production of interferon can be induced by enteroviruses containing only protein and RNA). The findings of Paucker and Henle (1958) suggested that only virus containing nucleic acid could induce interference and the results of Burke and Isaacs (1958a) indicated that treatment with UV light, which damaged influenza viral nucleic acid without significantly affecting its antigenic or neuraminidase activities, abolished its ability to produce interferon. These results focused attention on viral nucleic acid as the essential stimulus to make interferon. However, this still left unresolved the finding that both RNA and DNA viruses were able to stimulate cells to make interferon. An hypothesis was put forward that the essential stimulus to make interferon might be a nucleic acid that was “foreign” to the cell (Isaacs, 1981). This hypothesis was tested by treating chick and mouse cells with chick and mouse RNA and infecting them with vaccinia virus. It was found that the heterologous RNA showed a pronounced inhibition of virus growth, whereas homologous RNA showed much less inhibition or an absence of detectable inhibition. In addition, when 100 pg. of mouse RNA was incubated with chick cells, the cells washed, and then incubated with maintenance medium, the cells produced very small amounts of an antiviral substance that differed from the mouse RNA but resembled chick interferon in its properties (Rotem et d.,1963).* These results favor the hypothesis that production of interferon represents a response of cells to a foreign nucleic acid. One could speculate whether the interferon mechanism might play a role in other situations in which cells are exposed to a foreign nucleic acid. This could conceivably occur in the reaction of the body to skin grafts and possibly to tumor cells, which might both have nucleic acids that are foreign to the host Cells. Isaacs et aZ. ( 1963 ) have now produced additional evidence that production of interferon occurs as a response of cells to a number of non-viral “foreign” nucleic acids,

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Heller (1983) found that actinomycin D enhanced plaque production by a number of RNA viruses that multiplied in the cell cytoplasm. Such a result could be due to inhibition of the production or the action of interferon. This was investigated and actinomycin was found to have no measurable effect on the action of interferon. However, in doses similar to those that enhanced virus growth actinomycin strongly inhibited production of interferon. Besides accounting for the enhancing action of actinomycin, these results suggest that interferon production is under the control of DNA-dependent RNA synthesis. This implies that the genetic information for the production of interferon is normally present in cells and that the effect of virus infection is to release a mechanism that is normally suppressed. The findings of Levy and Baron (1963) were referred to already in Section V,D. Their results suggested that the target for interferon action is non-DNA-dependent RNA synthesis. What function this particular RNA synthesis might have is unknown, but its role in normal cells, in embryonic cells, and in tumor cells should be a rewarding subject for future research. VIII. INTERFERON AS A POSSIBLE THERAPEUTIC AGENT In addition to its antiviral action in vitro, administration of interferon in uivo has been found to induce varying degrees of protection in animals experimentally infected with certain viruses. Inhibition of the growth of vaccinia virus in the rabbit skin was reported by Lindenmann et uZ. (1957), Nagano and Kojima (1958), Isaacs and Westwood (1959a), and Andrews (1961), and in the rabbit cornea by Cantell and Tommila (1960). Some preliminary results on the effect of interferon on polyoma virus infection in hamsters were reported by Atanasiu and Chany ( 1980). Protection of mice against infection with Bunyamwera virus was found by Hitchcock and Isaacs (1960); this is of interest since it represents protection against a systemic virus infection. Kaplan et ul. (1982) found suggestive evidence that guinea pig interferon gave slight protection of guinea pigs against infection with rabies virus. The fact that interferon was active against a wide range of viruses, showed low toxicity and low antigenicity, and appeared to play a role in natural recovery from virus infections encouraged the idea that it might be developed as a possible therapeutic agent in man. With this end in view, a collaboration was set up in Great Britain between the Medical Research Council and three pharmaceutical firms-Glaxo, Wellcome, and I.C.I. Laboratories. This has resulted in the preparation of monkey interferon which was used in a first clinical trial. The trial carried out was an attempt to see whether interferon would influence the effects of primary vaccination in man. Volunteers were

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inoculated intradennally with interferon or control material given in coded form. The following day the volunteers were vaccinated at both sites and the vaccinia takes read “blind,” i.e., with no knowledge as to which site had received interferon or control material. Interferon was found to produce a highly significant degree of protection (Scientific Committee on Interferon, 1962). Some interferon was also used to treat a small number of patients with primary vaccinia1 keratitis and although this was not a controlled trial encouraging results were reported (Jones et al., 1962). Further trials of interferon in local v i r u s infections, i.e., virus infections of the eye and in common colds, are now in progress. Whether interferon will be found to benefit these conditions in a practical way remains to be determined. Possibly a sounder approach may eventually be to try to understand the nature of the stimulus to make interferon, so that it might prove possible to stimulate the natural resistance of individuals to virus infections.

REFERENCES Allison, A. C. (1961). Virology 15, 47. Andrews, H. D. (1961). Brit. Med. J . I, 1728. Atanasiu, P., and Chany, C. (1960). Compt. Rend. Acad. Sci. 251, 1687. Bader, J. P. ( 1962). Virology 16, 436. Bang, F. B., and Gey, G. 0. (1952). Bull. Johns Hopkins Hosp. 91, 427. Bang, F. B., Gey, G. O., Foard, M., and Minnegan, D. (1957). Virology 4, 404. Baron, S., and Isaacs, A. (1961). Nature 191, 97. Baron, S., and Isaacs, A. (1962). Brit. Med. J . I, 18. Baron, S., Porterfield, J. S., and Isaacs, A. (1961). Virology 14, 444. Bellett, A. J. D., and Cooper, P. D. (1959). J. Gen. Microbiol. 21, 498. Beveridge, W. I. B., and Bumet, F. M. (1946). Med. Res. Council Spec. Relit. Ser. 256. Burke, D. C. (1961). Biochetti. J. 78, 556. Burke, D. C., and Isaacs, A. (1958a). Brit. J. Expll. Pathol. 39, 78. Burke, D. C., and Isaacs, A. (1958b). Brit. J . Exptl. Pathol. 39, 452. Burke, D. C., and Isaacs, A. (1960). Acta Virol. (Prague) 4, 215. Cantell, K. (1961a).Arch. Ces. Virusforsch. 10, 510. Cantell, K. (1961b). Adoan. Virus Res. 8, 123. Cantell, K., and Tommila, V. (1960). Lancet ii, 688. Cantell, K., Skurska, Z., Paucker, K., and Henle, W. (1962). Virology 17, 312. Chany, C. ( 1961). Virology 13, 485. Cooper, P. D., and Bellett, A. J. D. (1959). J . Cen. Microbiol. 21, 485. De Maeyer, E., and De Maeyer, J. (1963). Nature 194,724. De Maeyer, E., and De Somer, P. (1962). Nature 194, 1252. De Maeyer, E., andEnders, J. F. (1963).In press. Denys, P., Jr., and Prinzie, A. (1962). Virology 17, 216. De Somer, P. (1962). Proc. Roy. SOC. Med. 55, 726. De Somer, P., Prinzie, A., Denys, P., and Schonne, E. (1982). Virology 16, 63. Dint-, Z. (1960). Acta Pathol. Micmbbl. S c u d . 49, 270.

36

ALICK ISAACS

Dinter, Z., and Philipson, L. (1962). Proc. SOC. Exptl. Bfol. Med. 109, 893. Enders, J. F. (1960). Trans. Studies Coll. Phystcians Phtlu. 28, 68. Fazekas de St.Groth, S., and Edney, M. (1952). J. lmmunol. 69, 160. Finter, N. (1962). Personal communication. Friedman, R. M., Baron, S., Buckler, C. E., and Steinmuller, R. I. (1962).J. Exptl. Med. 118, 347. Frilton, F., and Armitage, P. (1951). J. Hyg. 49, 247. Card, S . (1944). Acta Med. Scand. 119, 27. Gifford, G. E. (1903a). In preparation. Gifford, G. E. (1963b). To be published. Gifford, G. E., and Blakey, B. R. (1959). Proc. SOC. Exptl. B b l . Med. 102, 268. Gifford, G. E., and Heller, E. (lQ63).To be published. Gifford, G. E., Toy, S. T., and Lindenmann, J. (1963). Virology 19, 294-301. Glasgow, L. A., and Habel, K. (1902). J. Exptl. Med. 115, 503. Green, M., Henle, G., and Deinhardt, F. (1958). Virology I, 206. Gresser, I. (1961a). Proc. SOC.Exptl. Blol. Med. 108, 303. Gresser, I. ( l w l b ) . Proc. SOC. Exptl. Biol. Med. 108, 799. Gresser, I. (1961~).Proc. Natl. Acad. Sci. U.S 47, 1817 Gresser, I., and Enders, J. F. (1962). Vtrology 18, 428. Grossberg, S. E., and Holland, J. J. (1961). Federation Proc. 20, 443. Grossberg, S. E., and Holland, J. J. (1962). J. Immunol. 88, 708. Heller, E. (1903). Bbchem. J. 87, 18P. Henderson, J. R., and Taylor, R. M. (1961). Vtrology 13, 477. Henle, W. (1950). J. lmmunol. 84, 203. Henle, W., Henle, G., Deinhardt, F., and Bergs, V. V. (1959). J. Exptl. Med. 110, 525. Hitchcock, G., and Isaacs, A. (1960). Brit. Med. J. II, 1268. Hitchcock, G., and Porterfield, J. S. (1961). Virobgy 13, 363. Ho, M . (1961). Proc. SOC.Exptl. B b l . Med. 107, 039. Ho, M. (1902a). Vlrobgy 17, 262. Ho, M. (1962b). New Engl. J. Med. 266, 1258. Ho, M. (1062~).Intern. Congr. Mfcrobbl., Bth, Montreal, Abstrs., p. 101. Ho, M.,and Breinig, M. K. (1962). J . Zmmunol. 89, 177. Ho, M.,and Enders, J. F. (1Q59a).Proc. Natl. Acad. Sd. U.S. 45, 385. Ho, M., and Enders, J. F. ( 1959b). Virology 9, 446. Isaacs, A. (1959). Symp. SOC. Gen. bkrobiol. 9,102. Isaacs, A. (l960a). Symp. Sect. M4cpbioZ. N.Y.Acad. Med. 10, 182. Isaacs, A. (1960b). Virology 10, 144. Isaacs, A. (1961). Nature 192, 1247. Isaacs, A., and Baron, S . (1960). Lahcet ii, 946. Isaacs, A., and Burke, D. C . (1958). Nature 182, 1073. Isaacs, A,, and Fulton, F. (1953). J. Gen. Mlcrobbl. Q, 132. Isaacs, A,, and Hitchcock, G. (1960). Lancet ii, 69. Isaacs, A., and Lindenmann, J. (1957). Proc. Roy. SOC. Ser. B 147,258. Isaacs, A., and Westwood, M. A. (1959a). Lancet ii, 324. Isaacs, A,, and Westwood, M. A. (195913). Nature 184, 1232. Isaacs, A., Lindenmann, J., and Valentine, R. C. (1957). Proc. Roy. SOC. Ser. B 147, 268. Isaacs, A., Burke, D. C., and Fadeeva, L. (1958). Brit. J. Erptl. Pathol. 39, 447. Isaacs, A,, Klemperer, H. G., and Hitchcock, G. (1961a). Virology 13, 191.

INTERFERON

37

Isaacs, A., Porterfield, J. S., and Baron, S. (1961b). Virology 14, 450. Isaacs, A., Rotem, Z., and Cox, R. A. (1963). Luncet ii, 113. Jacob, F. (1959).Hnruey Lectures Ser. 54, 1. Jones, B. R., Galbraith, J. E. K., and Al-Hussaini, M. K. (1962). Lancet i, 875. Kaplan, M. M., Cohen, D., Koprowski, H., Dean, D., and Ferrigan, L. (1962). Bull. World Heulth Organ. 26, 765. Kilbourne, E. D., Smart, K. M., and Pokomy, B. A. (1961). Nature 190, 650. Lampson, G. P., Tyttell, A. A., Nemes, M. M., and Hilleman, M. R. (1963). Proc. SOC. Exptl. Biol. Med. 112, 468. Lennette, E. H., and Koprowski, H. (1946). J. Exptl. Med. 83, 195. Levy, H. B., and Baron, S. (1963). To be published. Levy, H. B., Glasgow, L. A., and Baron, S. (1962). Intern. Congr. Microbiol., 8th, Montreal, 1962, Abstrs., p. 83. Lindenmann, J. (1960). Z. Hyg. Infektionskrunkh. 146, 287. Lindenmann, J., and Gifford, G. E. (1963). Virology 19,302. Lindenmann, J., Burke, D. C., and Isaacs, A. (1957). Brit. J . Exptl. Puthol. 38, 551. Link, F., and Raus, J. ( 1961). Nature 192,478. Magee, W. E., and Sagik, B. P. (1959). Arch. Biochem. Biophys. 82, 340. Matumoto, M., Nishi, I., and Saburi, Y. (1959). Compt. Rend. SOC. B i d . 153, 1845. Mayer, V. (1962). Actu Virol. (Prague) 6, 317. Mayer, V., Sokol, F., and VilEek, J. (1961). Actu Virol. (Prugue) 5, 284. Mayer, V., Sokol, F., and Vilcek, J. (1962). Virology 16, 359. Mercer, C. K., and Mills, R. F. N. (1960). J. Gen. Microbiol. 23, 253. Mosley, J. W., and Enders, J. F. (1962). Virology 17, 252. Nagano, Y., and Kojima, Y. (1954). Compt. Rend. SOC.Biol. 148, 1700. Nagano, Y., and Kojima, Y. (1958). Compt. Rend. SOC. Biol. 152, 1627. Nagano, Y., and Kojima, Y. (1960). Compt. Rend. SOC. BWZ. 154, 2172. Nagano, Y., and Kojima, Y. (1961). Compt. Rend. SOC. Biol. 155, 1183. Niven, J. S. F., Armstrong, J. A., Balfour, B. M., Klemperer, H. G., and Tyrrell, D. A. J. (1962). J . Puthol. Bucteriol. 84, 1. Qrskov, J., and Andersen, E. K. (1938).Acta Puthol. Microbiol. Scund. Suppl. 37, 621. Paucker, K.,and Cantell, K. ( 1962). Vtrology 18,145. Paucker, K., and Henle, W. (1958). Virology 6, 198. Paucker, K., Cantell, K., and Henle, W. (1962). Virology 17, 324. Pollikoff, R., Donikian, M. A., Padron, A., and Liu, 0. C. (1962). Proc. SOC. Exptl. Biol. Med. 110, 232. Porterfield, J. S. ( 1959). Lancet ii, 326. Porterfield, J. S. ( 1963). In “Techniques in Experimental Virology” (R. J. C. Harris, ed.), Academic Press, New York. In press. Porterfield, J. S., Burke, D. C., and Allison, A. C. (1960). Virology 12, 197. Postlethwaite, R. (1960). Virology 10, 466. Rotem, Z., and Charlwood, P. A. (1963). Nature 198, 1086. Rotem, Z., Cox, R. A., and Isaacs, A. (1963). Nature 197,584. Ruiz-Gomez, J., and Isaacs, A. (1963a). Virology 19, 1. Ruiz-Gomez, J., and Isaacs, A. (196313). Virology 19, 8. Huiz-Gomez, J., Rotem, Z., and Isaacs, A. (1963). To be published. Sawicki, L. ( 1961). Nature 192, 1258. Sawicki, L., Baron, S., and Isaacs: A. (1961). Lancet ii, 680. Schlesinger, R. W. ( 1959). In “Viral and Rickettsia1Infections of Man” (T. M. Rivers and F. L. Horsfall, eds. ), pp. 145-155. Pitman Med. Publ., London.

38

ALICK ISAACS

Scientific Committee on Interferon. ( 1962). Lancet i, 873. Sellers, R. F. (1983). Nature 198, 1228. Sellers, R. F., and Fitzpatrick, M. (1962). Brit. J . Erptl. Pathol. 43, 674. Sueltenfuss, E. A., and Pollard, M. (1963). Science 139,595. Sutton, R. N. P., and Tyrrell, D. A. J. (1961). Brit. J. Exptl. Pathol. 42, 99. Thiry, L. (1962). Intern. Congr. Microbiol., 8th, Montreal, 1962. Abstrs., p. 85 and personal communication. Tyrrell, D. A. J. (1959). Nature 184, 452. VilEek, J. (1960). Nature 187, 73. VilEek, J. ( 1961). Acta V i d . (Prague) 5, 278. VilEek, J. ( 1962). Acta Virol. (Prague) 6, 144. Vilcek, J. ( 1963). Acta Virol. (Prague) 7, 107. VilEek, J., and Rada, B. (1962). Acta V i d . (Prague) 6, 9. Vogt, M.,Dulbecco, R., and Wenner, H. A. (1957). Virology 4, 141. von Magnus, P. (1954). Advan. Virus Res. 2,59. Waddell, G. H. ( 1962). Ph.D. Thesis, Univ. of Miami. Wagner, R. R. (1960). Bacteriol. Rev. 24, 151. Wagner, R. R. (1961). Virology 13, 323. Wagner, R. R. (1962). Cold Spring Harbor Symp.Quant. Biol. 27, 349. Wagner, R. R., and Levy, A. H. ( 1960). Ann. N.Y. Acad. Scl. 88, 1308. Zemla, J., and Schramek, S. (1962a). Acta Virol. (Prague) 8, 275. Zemla, J., and Schramek, S . (1962b). Virology 16, 204. Zemla, J., and VilEek, J. ( 1961a). Acta Virol. (Prague) 5, 129. Zemla, J., and VilEek, J. (1961b). Acta Virol. (Prague) 5, 367.