The Action of Normal Mouse Serum on Influenza Virus

Transcription

1 CHU, C. M. (1951). J. gen. Microbiol. 5, The Action of Normal Mouse Serum on Influenza Virus BY C. M. CHU The World Influenza Centre, National Institute for Medical Research, London, N. W.7 SUMMARY: Normal mouse serum contains a haemagglutination inhibitor and a neutralizing factor for influenza virus. The inhibitor and the neutralizing factor are heat-labile, can be destroyed by a crude filtrate of Vibrio cholere and are much more active towards unadapted virus than towards mouse-adapted virus. The inhibitor enters into stable combination with the virus in the presence of Ca ions. It is adsorbed by a large amount of unadapted virus, even after the haemagglutinin of the latter has been destroyed by heating at 60 for 15 min. Three strains of freshly isolated influenza B virus tested were not significantly neutralized by normal mouse serum. By growing two strains of unadapted influenza A virus in eggs in the presence of normal mouse serum, two variants which are resistant to the neutralizing action of mouse serum were produced. It appears that this type of variation also occurs when an unadapted virus is passed in mice. The theoretical implication of this type of variation in response to a normal host component and its relationship to the mouse-pathogenicity of influenza virus are discussed. The presence of haemagglutination inhibitor for influenza virus in normal human and animal sera has been described by various workers (Hirst, 1942, 1948; Francis, 1947; Anderson, Burnet & Stone, 1946; McCrea, 1946; Burnet & McCrea, 1946; Friedewald, Miller & Whatley, 1947). Burnet & McCrea (1946) found that normal ferret serum possessed a small inactivating power against one strain of influenza A virus (BEL) both in eggs and in mice. Smith & Westwood (1949) found that normal guinea-pig serum reduced the infectivity of PR 8 virus in mice. Typical Francis inhibitors, i.e. inhibitors present in normal tissues, secretions and sera resembling the inhibitor in normal human serum originally described by Francis (1947), do not neutralize influenza virus in vivo. Therefore, other serum factors are probably responsible for the inactivating effect observed. McCrea (1948) suggested that at least two types of normal haemagglutination inhibitors have been observed : the Francis type of inhibitor and the non-specific inhibitor. While the present study was in progress, Ginsberg & Horsfall (1949a) reported the presence of a heat-labile inhibitory and neutralizing component against Newcastle disease, influenza and mumps viruses in the serum of man, rabbit, guinea-pig and mouse. Most of their experimental work was carried out with guinea-pig serum and only laboratory-adapted strains of influenza virus (PR 8 and Lee) were employed. Their results, however, left little doubt that the guinea-pig serum inhibitor with which theydealt was in many ways similar to the normal mouse serum inhibitor reported in this paper. The present study was initiated by the observation that unheated normal mouse serum (N.M.S.), while inhibiting to fairly high titre haemagglutination by strains of influenza A virus which had not been adapted to mice, was very

2 740 C. M. Chu much less inhibitory to the mouse-adapted strains. It was further observed that this differential inhibition in vitro was often accompanied by a similar differential neutralization in vivo. It seemed that a more thorough investigation of these phenomena might lead to a better understanding of the mechanism of mouse adaptation of influenza virus. Accordingly, studies were undertaken to investigate the properties of these normal serum factors and their mode of interaction with the virus. Experiments were also made to grow the virus in eggs in the presence of normal mouse serum and in this way two variants were produced which are resistant to neutralization by the normal serum factor. The results of these studies are described in this paper. For the sake of clarity, throughout this paper, the term inhibition or inhibitor refers to haemagglutination inhibition, and the term neutralization or neutralizing factor refers to inactivation of infectivity in vivo. The term adaptation or adapted, unless otherwise specified, always refers to mouse adaptation. MATERIALS AND METHODS Virus strains. Many strains of influenza virus, of both type A and type By were used in the present investigation. As far as they are known, the origin and past history of these strains will be indicated when they are first mentioned in the text. Thus, Barratt (A/England/l947/M14) means that this strain is a type A virus isolated in England in 1947 and has had 14 mouse passages in addition to passages in eggs. Allantoic fluids for haemagglutination and inhibition tests were stored at 4. Viruses for infectivity or neutralization tests were always stored over solid carbon dioxide. Normal mouse serum (N.M.S.). Healthy mice of Parkes stock of the National Institute for Medical Research, weighing g. were used throughout. Blood was collected by putting the mice under deep anaesthesia, opening the chest and severing the aorta just above the diaphragm. Sera from 40 or more mice were pooled. With such large pools, only slight variations in inhibitory and neutralizing activities were observed between the individual pools. Mouse serum often contains a small amount of normal agglutinin against fowl red cells, which was removed by absorption before each pool was stored. The serum was then filtered through a 0*75p. Gradocol membrane and stored in a -60 deep-freeze. Unless otherwise stated, serum was always used without prior heat inactivation. Haemagglutination and haemagglutination-inhibition tests. The technique used has been described in detail elsewhere (Chu, Andrewes & Gledhill, 1950). Tests were carried out at room temperature with 0.5 yo (v/v) fowl red cells. Eight partial agglutinating units of virus were used in the inhibition test. The virus dilution showing a partial agglutination pattern was taken as the haemagglutination titre. The highest serum dilution giving complete inhibition of agglutination was taken as the inhibition titre. Normal mouse serum in low dilutions often causes slipping of agglutinated red cells, presenting a false negative pattern which sometimes makes the placing of titre rather

3 Mouse Serum on influenza virus 741 difficult. This difficulty was overcome when, in addition to direct reading of the pattern, the racks were held at an angle of 45' for about 30 sec. and the flow of the red cell 'button' at the bottom of the tubes observed. Unagglutinated cells should flow freely and agglutinated cells, not at all. Cells in the tube corresponding to the inhibition titre usually flow slowly but less freely than the negative control cells. All titres are expressed in terms of the initial dilution of the materials concerned. Infectivity titration and neutralization test in eggs. The constant serum method was adopted. Equal volumes of undiluted N.M.S. and tenfold dilutions of virus in broth-saline were mixed and incubated at 4' for 1/2-1 hr. Volumes of fluid (0.2 ml.) were injected into the allantoic cavity of 9-11-day-old chick embryos, using 4 eggs/dilution. Following incubation at 35' for 3 days, some allantoic fluid was aspirated-off and tested. Presence of haemagglutinin was taken as evidence of virus multiplication. Control virus titrations were always set up at the same time, with broth-saline replacing N.M.S. The 50% infective dose (ID 50) was calculated according to Reed & Muench (1938). The number of ID50 neutralized by the undiluted serum will be referred to as ' neutralization index' which is calculated by dividing the ID 50 for the virus mixed with serum by the ID50 for virus control. Passage of virus in eggs with normal mouse serum. The same technique as used for the neutralization test was followed. There was usually considerable neutralization by N.M.S. on the first passage. Fluid from one egg at the highest positive dilution was picked at random for passage, again in mixture with N.M.S. Such passages were repeated four or five times. A parallel control line was carried in eggs with broth-saline replacing N.M.S. and passed at dilutions comparable to those of the experimental line. Infectivity titration in mice. Five 'Parkes' mice (12-15 g.) were used for each dilution. They were lightly anaesthetized with an ether-chloroform mixture and were inoculated intranasally with 0.05 ml. of virus. All mice dying within 14 days were autopsied as soon as possible; surviving animals were killed by gas and examined for lesions. Occasional mice dead from nonspecific causes were excluded from the analysis of results. EXPERIMENTAL Haemagglutination-inhibition of inftuenxa A strains by N.M.S. Inhibition by N.M.S. to moderate titres seems to be characteristic of all A-prime strains isolated and maintained in eggs since Practically equal titres were obtained whether active virus or virus heated at 56' for 30 min. in citrate-borate buffer (Stone, 1949a) was used in the test. On the other hand, all the mouse-adapted strains including WS, PR 8 and Shope 15 were inhibited to much lower titres and even then the inhibition usually took the form of partial agglutination in several consecutive serum dilutions (Table 1). Repeated passages of an unadapted virus in the allantoic cavity of chick embryos slightly increased its inhibition by N.M.S. ; thus A/Paris-1 /P.L./1949 after 3 egg passages was inhibited to 1/80, the same strain after 21 passages when

4 742 C. M. Chu tested at the same time was inhibited to 1/240. In another experiment, by passing each of the two mouse-adapted strains (Paris-1 /Ma4 and Barratt A/England/l947/M 14) in eggs for 12 consecutive passages, a partial reversal to inhibition by N.M.S. (inhibition titre 1/20) was effected. Table 1, Strain ws PR 8 Barratt Barratt Williams Williams Paris Paris Roma Roma Haemagglutination-inhibition of influenza A strains by normal mouse serum Year and place of isolation 1933, England 1934, Puerto Rico 1947, England 1947, England 1947, England 1947, England 1949, Paris 1949, Paris 1949, Rome 1949, Rome Mouse-adaptation Adapted Adapted Unadapted Adapted (M 11) Unadapted Adapted (M 17) IJnadapted Adapted (M 24) Unadapted Adapted (M9) Inhibition titre of I iormal mouse serum < 10 < < < Figures under Inhibition titre in this and subsequent tables indicate the reciprocals of serum dilution indicates partial inhibition at 1/10 and 1/20. Figures in parenthesis indicate the number of mouse passages. These strains were tested with allantoic fluids from the first subculture of mouse lung virus in eggs. Haemagglutination-inhibition of injuenza virus at diflerent levels of mouse adaptation Strain A/Roma-2/1949 was repeatedly passaged in mice. Subcultures of mouse lungs were made in eggs after the first, third and sixth passages and the allantoic fluids tested for inhibition by N.M.S. In order to determine more quantitatively the degree of agglutination-inhibition at each serum dilution, the photoelectric densitometer method of Hirst & Pickels (1942) was adopted in this experiment. The curves shown in Fig. 1 clearly indicate that as mouse passage of the virus A/Roma-2/1949 went on, inhibition by N.M.S. was progressively decreased and after 6 passages, only partial inhibition was demonstrated. Increase in pulmonary lesions was observed after the third passage and mice began to die after the sixth passage. Neutralization of injuenza A strains The results of inhibition and neutralization tests with the unadapted and adapted lines of Barratt and Paris-1 and with the unadapted line only of A/Nederland-l/1949 are shown in Table 2. The neutralization indices for the unadapted lines varied from to , depending on the strain used. Since Barratt has had many egg passages before and was therefore better adapted in eggs, this may partly explain the relatively low neutralization index obtained. In general, there is no quantitative correlation between the inhibition titres and the neutralization indices when different strains are compared. Mouse-adapted viruses, however, were neither appreciably inhibited nor significantly neutralized by N.M.S.

5 Mouse serum on influenza virus 743 Dilution of normal mouse serum Fig. 1. Haemagglutination-inhibition by normal mouse serum of Roma-2 virus at different mouse-passage levels. MO-virus before mouse passage. M1, M3, M6virus after 1, 3 or 6 mouse passages respectively and subcultured once in the allantoic cavity of chick embryos. Effect of physical and chemical agents on N.M.S. inhibitor and neutralizing factor (1) Heat. Both the inhibitory and neutralizing activities of N.M.S. were destroyed by heating the serum at 56" for 30 min. An example of such heat inactivation, tested with unadapted Barratt, is shown in Table 2. No loss of inhibitory or neutralizing activities was noticed within 6 months when a pool Table 2. Neutralization in egs of unadapted and mouse-adapted strains of injluenza A virus by normal mouse serum Normal mouse serum Unheated Heated Unheated Unheated Unheated Unheated Haemagglutination inhibition titre of Virus titre-log ID 50 A Log neutral- Virus N.M.S. Without N.M.S. With N.M.S. ization index Barratt unadapted Barratt < > 7-50 < 1-00 unadapted Barratt adapted Paris unadapted Paris > 7.00 < 1.50 adapted Nederland unadapted

6 744 C. M. Chu of N.M.S. was stored at -60" and tested at intervals. A pool of serum kept at 4" for one week was found to retain its inhibition titre; its neutralizing activity was not tested. A heat-labile inhibitor in rabbit serum which was inactivated by heating to 62" for 20 min. has been described by McCrea (1946). The work of Ginsberg & Horsfall(1949a) has already been referred to. (2) Adsorption with kaolin. In previously unpublished experiments, the present author found that kaolin selectively removed the non-specific inhibitor from rabbit serum without significantly affecting the antibody titre. The same technique was applied to treat N.M.S. as follows. Kaolin (0-lg.) and N.M.S. (0.1 ml.) were mixed and incubated in corked tubes for 30 min. at room temperature. Saline (0.9 ml.) was added and the diluted (1 /lo) mixture centrifuged. The clear supernatant fluid was tested for inhibition titre against the appropriate viruses. A pool of N.M.S. originally inhibiting unadapted Barratt to 1 /is0 was found to be free from inhibitor after such treatment. When a pool of mouse anti- PR 8 serum was similarly treated, its titre against PR 8 was reduced from 1/240 to 1/160, whereas its titre against Barratt was reduced from l/l60 to less than 1/10. (3) Antagonists of Ca ions. van der Veen & Mulder (1950) found that sodium citrate and potassium oxalate solutions used as diluent decreased the nonspecific inhibition titre of ferret serum. The same effect was observed when normal saline containing 1 yo of sodium citrate, potassium oxalate or sodium metaphosphate was employed as diluent in testing the N.M.S. inhibitor. Addition of 1 yo calcium chloride to citrate saline restored the inhibition titre. Saline containing 1 yo calcium chloride, however, did not further increase the titre of N.M.S. when an unadapted virus was used, nor was an adapted virus inhibited by N.M.S. when calcium saline was used as diluent instead of ordinary saline. A similar effect of Ca ion antagonists on the inhibition of Lee virus by normal guinea-pig serum was reported by Ginsberg & Horsfall (1949 a) who showed in addition that a partial dissociation of the virus-inhibitor complex could be obtained with citrate saline. The N.M.S. inhibitor, therefore, resembled both the ferret and guinea-pig inhibitors in requiring Ca ions for its combination with the virus. (4) Sodium periodate. The N.M.S. inhibitor was not inactivated by treatment with 0.05 M sodium periodate by the technique of Hirst (1948). The same treatment completely destroyed Francis inhibitor present in normal human serum when heated Lee virus was used as the indicator virus. (5) Action of V. cholerae Jiltrate and trypsin. V. cholerae filtrate was prepared as described elsewhere (Chu et al. 1950). The receptor-destroying enzyme (R.D.E.) activity was assayed according to the method of Burnet & Stone (1947). Semi-purified R.D.E. was prepared from the filtrate by adsorption at 0" on fowl red cells and subsequent elution at 37" in a special boric acid-borate buffer containing Ca ions (Burnet & Stone, 1947). By this means, the R.D.E. activity was concentrated 2-10 times, while the slight proteinase activity found in the crude filtrate was decreased below detectable levels. Undiluted N.M.S. was treated with an equal volume of the appropriate

7 Mouse serum on influenza virus 745 enzyme preparation at 37" for hr. In order to inhibit bacterial growth streptomycin was added to give 500 units/ml. The mixtures were then adsorbed three times at 0" with a large excess of washed fowl red cells. This adsorption served to remove any residual R.D.E. which, if present in any quantity, would interfere with the subsequent inhibition or neutralization test. The successful removal of R.D.E. was ensured by adding fowl red cells to samples of mixtures and testing their agglutinability by the appropriate virus after 1 hr. incubation. An R.D.E. control was included in each experiment, treated and tested in exactly the same manner. In no case was sufficient residual R.D.E. found after the adsorption to interfere with the test. The treated serum samples were then tested for haemagglutination-inhibition and neutralization in eggs. The results (Table 3) showed that the crude cholera filtrate destroyed the inhibitor and the neutralizing factor in N.M.S., whereas the semi-purified R.D.E. did not. It may be noted that the R.D.E. activity of the purified preparations was higher than that of the crude cholera filtrate, and since a calcium-borate buffer was used as diluent, the optimal ph and ionic conditions for R.D.E. action have been provided. Table 3. Effect of V. choleraefiltrate on the inhibitor and mutralizing factor of normal mouse serum f Treated N.M.S. Serum treated R.D.E. activity Haemagglutination Log neutralization with units /ml. inhibition titre index V. cholerae filtrate 2560 < V. cholerae filtrate absorbed with red cells Po <5 nt R.D.E. eluate ) 3.50 Broth Treated sera tested against unadapted Paris-1. nt used here and in subsequent tables denotes not tested. Since the crude cholera filtrate used contained some proteinase, the possibility that the latter might be responsible for the inactivating effect was next investigated. Two preparations of trypsin, a semi-purified Fairchild injection trypsin, and a crystalline preparation (kindly provided by Dr A. S. McFarlane of this Institute) were tested, using the same technique as for the cholera enzyme. Under the experimental conditions, Fairchild injection trypsin, containing 100 ' trypsin units '/ml. decreased the titre of N.M.S. inhibitor at least eightfold, but 0-2 % crystalline trypsin had no effect at all. It is clear that neither R.D.E. nor trypsin itself acted on the N.M.S. inhibitor, but an unidentified active agent was present in both crude enzyme preparations. Mechanism of virus-inhibitor reaction The inhibition titre of N.M.S. is dependant on the amount of virus employed; within the limits of 4-16 agglutinating units, the relationship is A \

8 746 C. M. Chu practically linear. Outside this range, the inhibition curve deviates from a straight line, the titre being unduly high with 2 units of virus and unduly low and incomplete with 32 or more units of virus. The inhibition titre was increased by twofold if the serum-virus mixtures were incubated at room temperature for 1 hr. before the addition of red cells. Incubation beyond 1 hr. did not further increase the titre appreciably. Inhibition was very much decreased and incomplete at 4". Approximately the same titres were obtained when guinea-pig red cells were used instead of fowl cells; with human red cells, however, the titre was decreased fourfold. Two alternative hypotheses may be put forward to explain the mode of reaction between the virus and the inhibitor. It may be assumed that the reaction is essentially an enzymic one, and that the virus first enters into combination with the inhibitor and later destroys it. Alternatively, the virusinhibitor reaction may be an adsorption process involving relatively firm union with no dissociation under normal conditions. If the fist hypothesis is correct, one would anticipate that the virus would eventually be detected again after the inhibitor had been destroyed. This was not found experimentally. Actually, when serum and virus were mixed in such proportions that haemagglutination was just inhibited and the mixtures incubated at 37", no reappearance of haemagglutinin could be detected in samples removed and titrated at intervals up to 24 hr. This finding in itself favoured the second view which was further tested by the following experiment. Unit volumes of mouse-adapted or unadapted virus of equivalent haemagglutinin titre were added to equal volumes of N.M.S. and the mixtures incubated at 37" for 18 hr. The virus was then removed by filtration through Gradocol membranes of 150 mu. A.P.D. A preliminary experiment had shown that the inhibition titre of N.M.S. was not impaired by such filtration. The resulting filtrates were titrated for inhibitor content against an appropriate virus. When a large amount of virus (undiluted allantoic fluid with a haemagglutination titre of 1/1280) was used, the unadapted line of Barratt removed the inhibitor, whereas the mouse-adapted line of Barratt and PR 8 only slightly decreased the titre. Essentially the same results were obtained when unadapted and adapted Paris-1 were tested (Table 4). When, however, the serum was treated with a smaller amount of virus (Paris-1 allantoic fluid diluted 1/16) or with undiluted allantoic fluid previously adsorbed with red cells to remove all detectable haemagglutinin, only a small decrease of inhibitor titre was effected. Unadapted Paris-1 heated at 58" for 15 min. (which destroyed its eluting power from fowl red cells), or at 60" for 15 min. (which decreased its haemagglutination titre to less than 1/10) still removed the inhibitor; even virus heated at 70" for 15 min. retained some of its activity. It is clear that the N.M.S. inhibitor was only removed by the virus which it inhibited; a relatively large quantity of virus was required; and heating of the virus to temperatures which inactivated its other biological properties did not destroy its ability to combine with the inhibitor. These results give further support to the view that the removal of inhibitor is due to specific

9 Mouse serum on influenza virus 747 adsorption by the unadapted virus. Mouse-adapted virus probably has a changed surface configuration which prevents its effective combination with the inhibitor. Table 4. Adsorption of normal mouse serum inhibitor by unadapted and mouse-adapted influenza virus Inhibition titre of - adsorbed N. M. S. Adsorbing virus tested with A >- Mouse- Haemagglutina- Barratt Paris-1 Strain adaptation Treatment tion titre unadapted unadapted Paris-1 Unadapted Untreated 1280 <5 <5 Paris-1 Unadapted Heated 58' for 15 min nt <5 Paris-1 Unadapted Heated 60' for 15 min. < 10 nt <5 Paris-1 Unadapted Heated 70' for 15 min. ( Paris- 1 Unadapted Adsorbed with red cells < Paris- 1 Unadapted Diluted Paris-1 Adapted Untreated Saline nt = not tested. Variation of inhibitor content in individual mouse sera Since all the experiments hitherto reported were carried out with pooled sera from a large number of mice, it seemed interesting to investigate the variation of inhibition titres in the sera of individual animals. Forty healthy mice weighing g. were bled and their sera collected and tested individually against unadapted Paris-1. The distribution of inhibition titres was 4 mice at 1/20, 6 at 1/40, 19 at 1/60, 8 at 1/80 and 3 at 1/160. This gives a characteristic median distribution curve with about 50% of mice giving a titre of 1/60. Haemagglutination-inhibition and neutralization of influenza B strains Three strains of influenza B virus isolated in 1949 and 1950 were tested. They were moderately inhibited by N.M.S. in haemagglutination tests but none of them was significantly neutralized in eggs (Table 5). All three strains were initially isolated and maintained in eggs; B/London-5/1949 had had 8, and B/London-6/1950 had had 3, egg passages. Yet they were pathogenic to Table 5. Haemagglutination inhibition and neutralization in eggs of injeuenza B strains by normal mouse serum Haemagglutination Virus titre-log ID 50 inhibition titre, -A- - r Log neutralization Strain of N.M.S. Without N.M.S. With N.M.S. index Budapest London London Inhibition titre against unadapted Paris-1 included as control was None of the influenza B strains used in this experiment has been passed in mice.

10 748 C. 2M. Chu mice when allantoic fluid viruses were inoculated. The lethal dose (LD50) was characteristically low, about 10-1 or Dead mice showed pulmonary lesions typical of influenza infection. Inhibitors in other animal species Only limited data are available on inhibitors of A-prime virus strains in the serum of other animals. Normal human serum does not inhibit A-prime virus to any appreciable titre, usually less than 1/10. Other sera tested included those from sheep, ferret, rabbit, guinea-pig, hamster and cotton rat (Table 6). Hamster serum had the lowest inhibition titre. The other sera generally inhibited unadapted Barratt more than adapted Barratt, but the difference was not so marked as in the case of mouse serum. The sheep serum inhibitor was greatly diminished by heating at 56" for 30 min., and was readily destroyed by cholera filtrate. Table 6. Haemagglutination-inhibition of Barratt strains by unheated sera of diferent animal species Inhibition titre tested with A r \ Serum Unadapted Barratt Adapted Barratt Ferret Mouse 60 < 10 Rabbit Guinea-pig Hamster 10 < 10 Cotton rat 20 < 10 Of particular interest was the inhibitor present in normal ferret serum, partly because of its resemblance in some respects to the N.M.S. inhibitor, partly because of its practical importance when ferret anti-sera are used in the study of influenza virus. Like N.M.S., the inhibition titre of ferret serum was greatly decreased when citrate or oxalate saline was used as diluent (van der Veen & Mulder, 1950). The ferret serum inhibitor was only partially inactivated by heating at 56' for 30 min.; this may be due to the presence of more than one inhibitor. Treatment with cholera filtrate alone, instead of destroying the inhibitor, actually slightly enhanced its titre. After such treatment, however, the inhibitor could be completely inactivated by heating at 56" for 30 min. The mechanism of this inactivation by the combined action of cholera filtrate and heat is obscure, but since this procedure did not appear to affect to any appreciable extent the antibody of immune ferret serum, it has been applied empirically to the pre-treatment of ferret anti-sera for serological studies (van der Veen & Mulder, 1950; Chu et al. 1950). Treatment with purified R.D.E. followed by heating has no more inactivating effect on the ferret serum inhibitor than that achieved by heating alone. Variants of infuenza A virus produced by passage in egs in the presence of N.M.S. Two strains of influenza A virus, Paris-1 and Nederland-1, were employed. Both strains were isolated in 1949 and belonged to the A-prime group and

11 House serum on influenza virus 749 were antigenically indistinguishable from each other (Chu et al. 1950). Paris-1 had had 1 amniotic and 25 allantoic passages before being used in the present experiment; Nederland-1 had had 2 amniotic and 2 allantoic passages. For the sake of simplicity, the parent strains will be referred to as Paris-1 Co and Ned-1 Co respectively. Viruses of the control series will be referred to as Cl, C2, C3, etc., denoting virus after 1, 2 or 3 passages respectively. Similarly, viruses passed with N.M.S. will be referred to as S1, S2, S3, etc. (1) Paris-1 variant. Data relating to the origin and characters of this variant are presented in Table 7. This strain grew well in eggs before being used in the present experiment and through 6 passages, both in the control and N.M.S. series, the haemagglutination titre did not undergo any marked change. The inhibition titres by N.M.S. also remained fairly steady in both series. Approximately equal ID50 were obtained when the two lines after the sixth passage were titrated in eggs. Both C6 and S6 were equally inhibited by ferret anti-paris-1 serum. Significant difference in neutralization by N.M.S. was, however, noticed between the two series even after a single passage; the neutralization index for Cl, being 4.17, and for Sl, being This difference became more marked when both C6 and S6 were passed once at and the subcultured fluids tested, being 4.23 and 0-83 respectively. The parent strain was quite non-pathogenic for mice, and as a rule, only very small lesions were produced, but with undiluted inoculum, an occasional mouse out of a group of five might die with complete pulmonary consolidation ; such an instance was illustrated in Table 7. After 3 passages, S3 gained appreciably in mouse-virulence over C3, but it may be noted that the ID50 of the former in eggs was also tenfold higher. This increased virulence was maintained but not further enhanced in S6. In the control line, however, C6 also killed half of the mice when undiluted fluid was inoculated. Both lines were then subcultured in eggs, using 10-8 dilution. The allantoic fluids after the first and second subculture were titrated again in mice. The N.M.S. line consistently killed mice up to 10-1 dilution and occasionally at whereas the control line killed irregularly and usually less than half of the mice inoculated. It may be noted that with the exception of the third passage material, all other preparations from the two series had comparable egg infectivity titres. All the dead mice referred to had complete or nearly complete pulmonary consolidation typical of influenza1 pneumonia. Attempts were made to pass into fresh groups of mice 10% (w/v) lung suspensions of mice which had died within 12 hr. after the inoculation of C6 or S6 materials, but only minute lesions and no deaths were produced at the two dilutions tested (undiluted and 10-2). It appeared, therefore, that the pneumonia produced was of the non-transferable type described by Sugg (1949). (2) Nederland-1 variant. Data relating to the origin and characters of this variant are shown in Table 8. The parent strain grew only moderately well in the allantoic cavity of chick embryos and produced no lesions or death in mice.

12 Table 7. Paris-l variant produced by passage in eggs with normal mouse serum? Passage No I Neutralization index log nt nt nt nt nt 4.23 Control series A Mouse titre Egg titre A log ID50 loo / /5 2/ \ \ N.M.S. series A > Mouse titre Neutralization Egg titre -A- -, indexlog log ID50 loo lo- I 2.00 nt nt 8.5 nt nt nt 8-75 < s e 7/ Mouse titre was expressed as No. dead/no. inoculated , = 6 experimental passages in eggs followed by one or two subcultures in eggs by passage at 10 -* dilution. nt = not tested.

13 Table 8. Nederland-l variant produced by passage in eggs with normal mouse serum Control series N.M.S. series A A r \ I \ Passage Haemagglutination titre Neutralization Egg titre Haemagglutination titre Neutralization Egg titre % No. (Inhibition titre) index log log ID50 (Inhibition titre) index log log ID cb (80) Q 1 80 GO < 5.00 cb 1 (60) (50) (40) (40) *96 (80) (100) (Z) (60) > (80) 320 (120) 300 (80) (G) 50 (lo) $ m s (120) (15) a * The titres given are those of individual eggs tested. Figures in parenthesis refer to inhibition titres by N.M.S. The first eggs were used for passage and other experiments. Underlined figures refer to haemagglutination with large clumps of red cells which could not be redispersed at 37O, a rough indication that most of the virus present failed to elute. 5+1,5+2=5 experimental passages in eggs followed by one or two subcultures in eggs by passage at dilution. The titres for 5+2 represent the geometrical mean of 6 egg fluids titrations done at 4O. E E

14 752 C. N. Chu Marked neutralization by N.M.S. was observed in the first passage. After 2 passages in the presence of N.M.S., three striking changes in character were observed. Firstly, the N.M.S. line consistently gave lower haemagglutination titres relative to the control line. This relatively low haemagglutinin content in the fluid was accompanied by low ID50 figures in eggs. This character was maintained when S 5 variant was passed at l0-3in the absence of N.M.S. Thus, in the second subculture, when the control and N.M.S. lines were each inoculated into 6 eggs and the fluids harvested after 72 hr. incubation were titrated at 4" (this temperature was used to keep elution to a minimum), the geometrical mean of haemagglutination titre for the control line was 1/2000 and for the N.M.S. line was 1/215. Secondly, one out of two S1 eggs and all subsequent eggs in the N.M.S. series yielded fluids which agglutinated fowl red cells into massive clumps. These clumps were not redispersed by tapping nor by prolonged incubation at 37'. An adsorption-elution experiment with the fifth passage material indicated that S5 was adsorbed to fowl red cells at 4O as usual, but only 2.5 yo of adsorbed virus was eluted in 2 hr. at 37" as compared with 100 yo elution of C5. This character was also maintained when S 5 was subcultured twice in eggs at dilution. Thirdly, as anticipated, increased resistance to neutralization by N.M.S. was noticeable after the first passage and was unmistakable after the second. Reduction of haemagglutination inhibition by N.M.S. was less striking, but S 5 was definitely less inhibited than the corresponding C 5. Since at the same time the variant appeared to have increased its affinity for fowl red cells, this decrease of inhibition titre may not have been entirely due to a genuine resistance to the inhibitor. The finding of a virus which in the active state failed to elute from red cells suggested that, like heated Lee virus, it might serve as a good indicator for Francis inhibitor. This expectation was confirmed experimentally. As inhibitor, a preparation of sheep salivary gland mucoid, obtained through the courtesy of Dr J. F. McCrea (Lister Institute of Preventive Medicine, Chelsea), was used in 1% concentration. It was found that active Ned-1 C 5 was only partially inhibited at 1/10, while active S 5 was inhibited to 1 /320. Both viruses were converted into indicators, i.e. became inhibited by the mucoid inhibitor by heating at 56" for 30 min. in citrate-borate buffer (Stone, 1949a). Burnet, McCrea & Stone (1946) found that different virus strains varied in the degree to which they were able to remove red cell receptors, and could be arranged accordingly into a 'receptor gradient'. A virus 'early' in the gradient removed cell receptors for the homologous virus, but still left receptors for any virus 'later' in the gradient. A virus late in the gradient removed receptors for the homologous virus and for viruses earlier in the gradient. Similarly, Stone (19494 found that different virus.strains could be arranged into an 'inhibitor gradient' according to the extent they were capable of destroying the inhibitor for the homologous and other strains. Isaacs & Edney (1950) demonstrated by limiting dilution technique a variant of

15 Mouse serum on influenza virus 753 Melbourne virus which occurred earlier in the receptor and inhibitor gradients than the parent virus. The variation in the haemagglutination and inhibitory character of Ned-1 variant suggested the possibility that its position in the receptor and inhibitor gradients might also be altered. The test for receptor gradient was carried out according to the technique of Burnet et al. (1946). Fowl red cells were incubated at 37" with undiluted Ned-1 C5 for 4 hr. when the red cells had been completely re-stabilized. The treated cells were then washed once and used for the titration of Ned-1 S5. The same agglutination titres were obtained with treated cells and control cells, indicating that C5 had removed the receptors for the homologous virus but not for S5. Unfortunately, the reverse reaction could not be tested since S5 did not elute from fowl red cells. An experiment performed by Dr A. Isaacs showed that fowl red cells treated with different concentrations of R.D.E. until they are no longer agglutinated by C5 were still agglutinated by S 5. The inhibitor-destroying test was carried out by a slight modification of the method of Burnet (1949). Samples of 1 % sheep salivary gland mucoid were incubated with 100 agglutinating units each of C5 and S5 viruses for 18 hr. at 37'. The mixtures were then heated at 65" for 30 min. and the residual inhibitory titre estimated. It was found that C5 completely destroyed the inhibitor for the heated homologous virus, but not for heated S5 or heated Lee. Destruction of inhibitor by S5 was not complete in 18 hr., suggesting a very slow enzymic action which may be related to the failure of S5 to elute from fowl red cells. However, the inhibitory titre was decreased by fourfold for heated S 5, sixteenfold for heated C 5 and eightfold for heated Lee. From the foregoing experiments, it may be concluded that Ned-1 variant occurred 'later' than the control line in fowl cell receptor and sheep salivary gland inhibitor gradients. Tests for mouse-pathogenicity of Ned-1 variant were hampered by its relatively poor growth in the allantoic cavity. An attempt was made to concentrate the virus by centrifugation at 18,000 r.p.m. for 1 hr., but the deposited virus seemed to have been partly inactivated so that in spite of its higher haemagglutination titre, the ID50 for eggs was about the same as that of straight allantoic fluid. However, one experiment with S3 (-log ID50 in eggs 6.63) and another with S4 subcultured once (-log ID50 in eggs 7.00) only produced very small lesions and no deaths in mice within the tested range of undiluted to dilutions. When examined by electron microscopy after adsorption on laked fowl red cells, Ned-1 variant showed many filamentous as well as rod-like and spherical forms. The picture was indistinguishable from that of control virus. The antigenic relationship between the parent and variant lines of Ned-1 as shown by the anti-haemagglutinin test is as follows: GMV4 Serum '< Virus Ned-1 parent Ned-1 variant Anti-Ned-1 parent Anti-Ned-1 variant I0

16 754 C. N. Chu The characteristic feature was that whereas the ferret anti-serum against the parent strain inhibited the homologous virus to a titre 10 times higher than the variant, the anti-serum against the variant inhibited both to about equal titres. The two lines are antigenically closely related but not identical. DISCUSSION The differences between N.M.S. inhibitor and Francis inhibitor may be conveniently summarized as follows: Normal mouse serum inhibitor Thermo-labile. Inhibits active as well as heated virus to approximately equal titres. Inhibits unadapted (mouse) virus much more than mouse-adaptedvirus. Destroyed by crude cholera filtrate, but not by purified R.D.E. Not destroyed by periodate. Forms stable combination with active virus but is not destroyed by it. May be related to a factor which neutralizes unadapted virus in eggs. Francis inhibitor Thermo-stable Inhibits heated virus to much higher titres than active virus. Inhibits both adapted and unadapted virus. Destroyed by crude and purified R.D.E. Destroyed by periodate. Forms transient combination with active virus and is subsequently destroyed by it. No neutralization of virus in eggs, The guinea-pig serum inhibitor studied by Ginsberg & Horsfall (1949 a) closely resembles N.M.S. inhibitor. Thus, they are both heat-labile, inactive in the absence of Ca ions and enter into stable combination with the virus. These authors, however, also found their PR8 and Lee strains appreciably inhibited by unheated mouse serum. This discrepancy from the results here reported may be partly due to technical differences, but more likely is due to strain differences, as it has been shown that repeated passages of a mouseadapted strain in eggs may partly restore its property of inhibition by N.M.S. The same authors found that their Lee strain was not neutralized in eggs by mouse serum, but both their PRS and Lee strains were neutralized by guineapig serum, This finding would suggest a subtle difference between the neutralizing factors present in the two species and that a strain rendered resistant to N.M.S. by mouse-adaptation may still be neutralized by guineapig serum. Both the inhibitor and neutralizing factors occur in the mouse serum; both are active against the unadapted virus and much less so against the adapted virus; both are destroyed by heating at 56" for 30 min. and by cholera filtrate. These findings strongly indicate that the two factors are closely related if not identical. On the other hand, the absence of consistent positive correlation between the inhibitor titres and neutralization indices of various strains

17 Mouse serum on influenza virus 755 apparently contradicts such an assumption. It may be pointed out, however, that a strict comparison of results obtained by two such different techniques can lead to fallacious conclusions. The result obtained with either technique was inherently dependant on the relative affinity between virus and inhibitor, and virus and red cells or susceptible cells lining the allantoic cavity. Furthermore, a preparation consisting of 90% unadapted and 10% adapted virus will behave like an unadapted virus in the haemagglutination-inhibition test and more like an adapted virus in the neutralization test. Further evidence will be needed to decide the precise relation between the inhibitory and neutralizing factors. Inhibition and neutralization by N.M.S. appear to be characteristic of all unadapted influenza A-prime strains tested. None of the influenza B strains tested was significantly neutralized in eggs, although they were inhibited in vitro. It may be recalled that freshly isolated influenza B strains differ in a number of characters from the A-primes; they do not show any clear-cut O>D change (Burnet, Beveridge & Bull, 1944); they are often mousepathogenic to low titre without any history of mouse-adaptation and do not show filamentous forms characteristic of all freshly isolated A-prime strains (Chu, Dawson & Elford, 1949). Only a few unadapted A strains isolated in 1943 and antigenically related to PR8 have been examined by these criteria: the results obtained so far indicate that they are more akin to A-prime than to B strains. The experimental evidence on the correlation between the mouse-pathogenicity of a strain and its susceptibility to haemagglutination-inhibition and neutralization by N.M.S. may be summarized here. (1) All mouse-pathogenic strains, whether occurring naturally (such as the B strains) or produced by adaptation are resistant to neutralization. Their reactions to haemagglutination-inhibition were more irregular. As a rule, all freshly mouse-adapted viruses have shown significant and often striking resistance to inhibition. (2) The converse, however, does not hold true. Ned-1 variant, for instance, which was resistant to the neutralizing factor and was significantly less inhibited by N.M.S., did not show any mouse-pathogenicity. (3) The slightly enhanced mouse-pathogenicity of Paris-1 variant cannot be definitely correlated with the simultaneously developed resistance to neutralization by N.M.S. The facts that the control line in the course of egg passages has also gained some mouse-pathogenicity and that the pneumonia was only produced with very low virus dilutions and was of the non-transferable type are obviously against the suggestion that a genuine mouse-adapted virus has been produced. Isaacs & Edney (1950) have succeeded by a limiting dilution technique in isolating a variant from Melbourne virus which appeared to show lower mouse-pathogenicity than the parent virus. It seems likely that the process of selection at high dilution inherent in the method adopted here may have quite incidentally picked out natural variants having slightly greater pathogenicity for mice. On the foregoing evidence, it may be concluded that increased resistance

18 756 C. N. Chu to N.M.S. accompanies the process of mouse-adaptation and can be produced in eggs by passage with N.M.S. Such increased resistance may conceivably favour the multiplication of the virus in the mouse lung by overcoming a normal defence mechanism of the host, but it does not appear to be the sole or even primary factor in determining the mouse-pathogenicity of a virus. This phenomenon is nevertheless of considerable interest from the viewpoint of virus variation. Ginsberg & Horsfall(l949 b) reported the isolation of a variant of mumps virus resistant to the action of Bacillus friedldnder polysaccharide by growing the virus in the presence of this chemical. Isaacs & Edney (1950) found that by passing Melbourne virus in the presence of increasing concentrations of homologous anti-serum, a mutant form was obtained which was more resistant to neutralization by anti-serum in 0210 than the original virus. These variants, however, were only the result of special laboratory manipulations. To the author s knowledge, the N.M.S. resistant variant is the only case of variation in an animal virus which was produced in response to a normal host component and which in fact invariably occurred when the virus was adapted to this particular host. The mechanism by which the concomitant changes (such as loss of eluting power, slow enzymic activity, slower or poorer growth in the allantoic cells, shift in the gradient position and slight antigenic alteration) were brought about in Ned-1 variant is obscure. They may be independent characters selected at random. The fact that some of these characters can be readily measured by simple techniques suggests that this strain may be useful in further studies on the variation of influenza virus. I wish to express my thanks to Dr C. H. Andrewes for his interest and criticisms in this work, and to Mr Eric Owen for his technical assistance. REFERENCES ANDERSON, S. G., BURNET, F. M. & STONE, J. D. (1946). A modified Salk method for in vitro titration of influenza antibodies. Aust. J. exp. Biol. me&. Sci. 24, 269. BURNET, F. M. (1949). Specific relation between active virus and the corresponding indicator strains in studies of soluble inhibitors. Aust. J. exp. Biol. med. Sci. 27, 575. BURNET, F. M., BEVERIDGE, W. I. B. & BULL, D. R. (1944). Study of a strain of influenza B virus isolated by chick embryo inoculation. Aust. J. exp. Biol. med. Sci. 22,9. BURNET, F. M. & MCCREA, J. P. (1946). Inhibitory and inactivating action of normal ferret sera against an influenza virus strain. Aust. J. exp. Biol. med. Sci. 24, 277. BURNET, F. M., MCCREA, J. F. & STONE, J. D. (1946). Modification of human red cells by virus action. 1. The receptor gradient for virus action in human red cells. Brit. J. exp. Path. 27, 228. BURNET, F. M. & STONE, J. D. (1947). The receptor-destroying enzyme of V. cholerae. Aust. J. exp. Biol. med. Sci. 25, 227. CHU, C. M., ANDREWES, C. H. & GLEDHILL, A. W. (1950). Influenza in Bull. W.H.O. 3, 187. CHU, C. M., DAWSON, I. M. & ELFORD, W. J. (1949). Filamentous forms associated with newly-isolated influenza virus. Lancet, i, 602.

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