Department of Microbiology, John Curtin School of Medical Research, Australian National University, Canberra, Australia. (Accepted 19 November 1967)

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1 J. gen. ViroL (I968), 2, Printed in Great Britain 385 Serological Relationships between the Neuraminidases of Influenza Viruses By C. K. J. PANIKER* Department of Microbiology, John Curtin School of Medical Research, Australian National University, Canberra, Australia (Accepted 19 November 1967) SUMMARY Serological relationships between neuraminidases of representative strains of influenza virus isolated between 193o and 1967 were studied, using rabbit antisera. The neuraminidases of type A strains formed three antigenic groups, sw, Ao-A I and A2, with antigenic drift within these groups. The neuraminidases of type B strains were unrelated to those of type A strains, and also showed antigenic drift. Four subgroups could be distinguished in haemagglutination-inhibition tests, sw, Ao, A I and A2. Antigenic drift occurred within each of these subgroups; antigenic changes in the neuraminidase and haemagglutinin occurred independently. The neuraminidases of different strains as well as variants of a single strain varied greatly in their heat stability, which had no direct relation to the antigenic changes or the time of isolation of the strains. Both the antigenicity and the enzymic activity of the neuraminidases of certain strains (NSW, WSE) were heat-labile and it was necessary to use freshly prepared virus suspensions for preparing antisera to their neuraminidases. INTRODUCTION There are at least two virus-coded subunits on the surface of the influenza virus particle; the haemagglutinin and the enzyme neuraminidase. The two subunits can be separated by a variety of physical and chemical methods (Mayron et al. I96I ; Wilson & Rafelson, 1963; Laver, I963; Seto, Drzeniek & Rott, 1966) and both are antigenic, either as components of the intact particle or in the isolated state. There is a considerable body of information about antigenic variation in the influenza virus group (Jensen, 1957; Francis & Maassab, 1965; Green et al. 1964) based on studies of the haemaggiutinin antigen. These studies have shown that 'immunological or antigenic drift' (Burnet, 1955) occurs within the various groups of influenza viruses A (Ao, A I, A2) and B. Neuraminidases from chick chorioallantois, Vibrio cholerae (Ada, Lind & Laver, I963), Sendai virus (Tozawa, Homma & Ishida, 1967), Newcastle disease virus and some strains of influenza viruses (Ada et al. 1963; Drzeniek, Seto & Rott, 1966) are immunologically distinct, but no systematic study has been reported on the serological relationships between the enzymes of different strains of influenza viruses. In the present investigation the serological cross-reactions between the neuraminidases of representative strains of influenza viruses were measured in parallel with crossreactions among their haemagglutinins. The heat stability of the neuraminidase, as an enzyme and as an antigen, was also studied in some strains of influenza virus. * Present address: Department of Bacteriology, Medical College, Calicut, Kerala, India. 25-2

2 386 C. K. J. PANIKER METHODS Influenza virus strains. The strains used and the years of their isolation are listed below. Swine: I93O, strain 15 (Shope, 1931). Type Ao: 1933, WSE (Burnet, 1951). 1933, Nws (Stunt-Harris, I939). 1934, PR8 (Francis & MagiU, 1935). 1935, MEL (Burnet, 1935). 1941, HOL (obtained from the Commonwealth Serum Laboratories, Melbourne). 1942, BEL (Anderson & Burnet, 1947). 1943, CKS (Anderson & Burnet, 1947). Type A I: I946, CAM (Anderson & Burnet, 1947). 1947, FM I (Rasmussen, Stokes & Smadel, 1948). 1954, A I/QUEENSLAND/54 (obtained from the Commonwealth Serum Laboratories, Melbourne). 1956, At/WEST AUSTRALIA/56 (obtained from the Commonwealth Serum Laboratories, Melbourne). Type A2: 1957, A2/AA/23/57) obtained from the Strain Study Center, School of 1963, A2/AA/4/63 f Public Health, Ann Arbor, Michigan. Type A2 variants: R 15+; R 15- (Choppin & Tamm, 1959). Type A 2 recombinant x-7, which has the enzyme derived from the A 2 parent (R 15 +) and the internal antigen and the haemagglutinin from the Ao parent (NWS) (Laver & Kilbourne, 1966). Type B: I94O, LEE (Francis, 194o). 1953, B/MELBOURNE/53 (obtained from the Commonwealth Serum Laboratories, Melbourne). I962, B/TAIWAN/2/62 (Green et al. 1964) I967, B/ROM/67 (obtained from the World Influenza Centre, London). All strains except those separately indicated were from the collection maintained in the Department of Microbiology, Australian National University, Canberra. Strains of influenza virus types A and B were grown in the allantoic cavity of I I-dayold chick embryos. The infected allantoic fluids were centrifuged at low speed (2oo g for 30 min.) and the virus was concentrated by adsorption to and elution from chicken erythrocytes. The concentrated virus preparations were preserved with o.08 % sodium azide and stored at 4 ; these preparations were used for haemagglutination-inhibition tests. Virus preparations required for enzyme studies and as antigens for immunizing rabbits were prepared by differential centrifugation. Infected ahantoic fluids were clarified by low-speed centrifugation and then centrifuged in the cold at 53,000 g for 60 rain. to sediment the virus. The virus pellets were resuspended in calcium-magnesium saline (Fazekas de St Groth, Graham & Jack, 1958) to give about a xoo-fold volume concentration, snap frozen and stored at Haemagglutinin titration. Haemagglutinin (HA) was titrated in 0-25 ml. volumes in W.H.O. Perspex trays using o'5 % fowl erythrocytes. The end-point of the titration was read by interpolation between complete agglutination and no agglutination. One

3 Antigenic drift in influenza virus enzymes 387 HA unit was defined as that amount of virus giving partial agglutination (t5% dimers) with 0"25 ml. of 0"5 % fowl erythrocytes. Haemagglutination-inhibition (H1) test. All sera were inactivated to destroy nonspecific inhibitors of HA. The sera were heated at 560 for 30 min. with an equal volume of trypsin (Difco, 1:250, 4"0 mg./ml.). Three volumes of o.oii M-potassium periodate solution were then added and the mixture left on the bench for I5 min. Glycerol saline (1%) was then added to bring the serum dilution to I/2o. All sera were absorbed with fowl erythrocytes to remove agglutinins. HI was tested in perspex trays using 0"25 ml. volumes of serial twofold serum dilutions to which o'o25 ml. of 5 % fowl erythrocytes was added followed immediately by 4 HA doses of virus in o'o25 ml. The trays were shaken and readings taken 35 min. later. The HI titre of serum was expressed as the reciprocal of the serum dilution which inhibited three out of four HA doses of the virus. Pre-immunization samples of sera were always tested along with the respective antisera to verify that non-specific inhibitors of HA had been removed. Neuraminidase assays. Virus samples for neuraminidase assays (o.i ml.) were incubated at 35 for 3o min. with fetuin (I'25 mg.) and o.i M-sodium phosphate at ph 5"9 in a final volume of o.2 ml. The N-acetyl neuraminic acid liberated was assayed by the method of Warren 0959), except that the colour was extracted into n-butanol containing 5"0% (v/v) concentrated hydrochloric acid (Aminoff, I960. One unit of neuraminidase was defined as the amount liberating Io -9 moles of N- acetyl neuraminic acid per minute from fetuin under these conditions. Fetuin was prepared by the method of Graham (I960. Antineuraminidase assay. Titrations of antineuraminidase antibodies were made by preincubating influenza viruses (2 enzyme units in 0"05 ml.) with serial dilutions of heat-inactivated antiserum (o'05 ml. of serum heated at 560 for 30 min.) at 35 for 3 min. Pre-immunization samples of sera were also tested at the same dilutions simultaneously as the activity of neuraminidase is known to be influenced by the presence of normal serum (Ada et al I963). The percentage inhibition of neuraminidase was plotted against the serum dilutions, and a linear relationship was usually obtained between the two in the range of the test. The antineuraminidase titre of a serum was expressed as the reciprocal of the final dilution of serum in the reaction mixture inhibiting 50 % of the enzyme activity. Heat inactivation ofneuraminidase. Samples of virus containing two units of enzyme in o-i ml. volumes were heated in a water bath for varying periods and enzyme activity assayed. Samples were kept at o except during the period of heating. Inactivation was at 45 except where otherwise specified. Rabbit antisera. Viruses (50o0 HA units in 0"5 ml. saline) were inoculated into the marginal ear vein of adult rabbits, which were reinoculated after 40 days. A third dose of WSE 20 days after the second was required for production of anti-enzyme antibody. Sera were collected before immunization and I week after revaccination and stored without preservative at - I5. Two rabbits were vaccinated with each strain of virus. RESULTS Serological cross-reactions among neuraminidases The virus strains tested fell into four distinct serological groups (Table I). The neuraminidase of sw virus was neutralized only by the homologous antiserum and

4 * Titres represent the average reciprocal values for two different antisera. Figures in parantheses are the percentages of the homologous titre. t <, no inhibition or less than 50 % inhibition of neuraminidase at the lowest dilution of serum tested (I/IO initial dilution). NT, not tested. L~ OO OO O > Z r~ Table I. Serological cross-reactions of influenza virus neuraminidases Antigens A o strains A 1 strains A z strains B strains Rabbit antisera sw/3 O wse/33 PR 8/34 MEL/35 BEL/42 CAM/46 FM I/47 A I/WA/56 A 2/23/57 A 2/4/63 LEE/40 B/TAI/62 SW/ WSE] PR 8/ MEL/ HOL/ BEL] CKS/ CAM/ FM I/ A I/Q/ A I/WA/ A2/23/ A2/4/ LEE/ B/M/ B/TAI/ B/ROM/ x5" <t < < < < < < < < < < < < < < NT (ioo) < 62 < 40 < 40 < < < < < < < < NT < NT (ioo) (65) (65) < < o 112 ioo < < < < < NT < NT (ioo) (69) (48) (43) (28) (35) (21) < < i78 2x6 lo2 i3o < < < < < < NT < NT (82) (ioo) (47) (60) (44) (23) < < o x76 80 ioo < < < NT < NT (38) (41) (74) (ioo) (45) (57) (153) (34) (ii) < < o < < < NT < NT (io) (16) (40) (30) (29) (I00) (52) (35) (15) < < < < I00 84 I < < < NT < NT (I8) (I5) (I9) (50) (IOO) (I5) (9) < < < < 60 < I66 < < < NT < NT (36) (27) (48) (18) (13o) (loo) < < < < < < < < < < < goo 27 < NT < NT (ioo) (34) < < < < < < < -< < < < NT < NT (64) (IOO) < (87) (59) < < < < < < < < < < < < 200 (i0o) < 44 (22) < < < < < < < < < < < < < < < (IOO) 94

5 Antigenic drift in influenza virus enzymes 389 SW antiserum did not neutralize the enzyme of any other virus. The neuraminidases of human Ao and A I strains constituted a large inter-related group, the members of which showed sequential antigenic drift. The neuraminidase of WSE was a poor antigen and only very low titre sera could be obtained even after three intravenous doses of the virus. Although WSE antisera neutralized MEL and BEL enzymes, WSE enzyme was neutralized only by its own antiserum. Apart from WSE, the other members of this group showed strong cross-reactions. The two A2 strains tested cross-reacted with each other, but did not react with antisera to Ao or A I strains, nor did A2 antisera neutralize any but A2 enzymes. The two A2 strains, isolated at an interval of 6 years, showed evidence of antigenic drift. The four type B strains constituted a distinct group, showing antigenic drift within the group. There was no cross-reaction between the enzymes of types A and B strains. Table 2. HI and anti-neuraminidase titres of NWS (,40), R I5+ (.42) and x-7 (recombinant antisera against R I5+ and x-7 viruses) HI titre Antineuraminidase titre t ~ t Antisera g I5 + x-7 R I5 + x-7 NWS (A o) < < R 15+ (A 2) 1564 < x-7 (recombinant) < Apparent neutralization of neuraminidase by antihaemagglutinin When intact virus particles were used as the source of enzyme in cross anti-neuraminidase tests, as in this study, one course of error was the apparent neutralization of the enzyme by any cross-reacting antihaemaggiutinin antibody coating the viral surface and causing steric hindrance of enzyme action. This may have given rise to false-positive cross-reactions. To assess the maximum extent of this effect, antisera against NWS, R I5 + and recombinant x-7 viruses were tested for neutralization of g 15 + and x-7 enzymes (Table 2). Since x-7 virus has the haemagglutinin of NWS, it showed complete cross-reaction with NWS in HI test. As the enzymes of A2 and Ao strains are unrelated, x-7 enzyme should not be neutralized at all by ~ vcs antiserum, but it was seen that enzyme neutralization did occur to some extent. Antiserum to x-7 virus had a homologous HI titre of 2432 and antineuraminidase titre of Anti-Nws serum had an HI titre of 7680 and anti-enzyme titre of 344 with x-7 virus. Thus, even in this artificial situation where two viruses had identical haemagglutinins but different neuraminidases, the maximum error due to apparent neutralization of neuraminidase by cross-reacting haemagglutinin was less than Io %. Serological cross-reactions among haemagglutinins All antisera gave high homologous titres except those against type B strains (Table 3). The haemagglutinin of type A strains tested fell into four antigenic groups, sw, Ao, A1 and A2, though there was some cross-reaction between the groups, sw crossreacted with some Ao and A I strains, but the cross-reactions did not exceed 4 %. All Ao strains were inter-related and showed antigenic drift within the group. While Ao antisera cross-reacted only feebly with A I haemagglutinins, there was much greater cross-reaction between Ao haemagglutinins and A I antisera. CAM (A I) antiserum

6 * Titres represent the average reciprocal values for two different antisera. Figures in parentheses are the percentages of the homologous titre. t <, no inhibition of haemagglutination at the lowest dilution of serum tested (1/2o). NT, not tested. B strains tao O 2 Table 3. Serological cross-reactions between influenza virus in haemagglutination-inhibition tests Rabbit sw/ antisera 30 wse/33 PR8/34 MEL/35 SW/ * (loo) (4) (3) WSE/ (3) (IOO) (7) (3) PR8/ (2) (13) (IOO) (7) MEL/ (2) (1oo) BEL/ (2) (16) (61) (14) CAM/46 Io IOO (lo) (12) (3) rmh (3) (5) (4) (3) A I/WA/56 3o (I2) (3) (2) A2/23/57 < < 30 < (1) A2/4/63 < 26 < 20 (2) (1) LEE/40 < NT < NT B/TAI/62 < NT < Antigens A o strains A I strains A 2 strains AI/Q/ AI/WA/ A2/23/ A2/4/ HOL/4I BEL/42 CKS/43 CAM/46 FM1/ <t < < < < I0 < < (i) o4 48o (2) (1) (2) (1) 45 < (39) 5888 (IO0) lo56 (I8) (5) (2) (I3) (loo) (23) 38 < (I) 07) (loo) < < I (21) (25) < < < < < < < < < < NT NT NT NT < NT NT NT NT NT < < < 158 IOO < < IO IO IO o < 20 (6) (4) (9) (6) 464 (15) 3200 (loo) (i) < < (I0O) (73) < < (36) (loo) NT NT < NT NT NT < NT LEE] B/M] B/TAI] B/ROM/' (IOO) (54) (12) (17) oo (13) (59) (10o) (46)

7 Antigenic drift in influenza virus enzymes showed I2 and I3% cross-reactions with PR8 (Ao) and CKS (Ao) respectively and A I/WA/56 (A I) antiserum showed I2 % cross-reaction with wse (Ao). The haemagglutinins of A2 strains were antigenically very different from other type A strains, and the maximum cross-reactivity with other type A strains did not exceed 2%. The two A2 strains tested were closely related. There was no cross-reaction between the haemagglutinins of type A and B strains. All four type B strains tested cross-reacted in the HI test, and showed evidence of antigenic drift. LE~ antiserum showed only I2 and I7 % cross-reaction with B/TAI/62 and B/ROM/67 and B/TAI/62 antiserum only I3 % with L~E. 39 I Time (min.) Fig. ]. Inactivation of influenza virus neuraminidases at 45. O, B/TAt]62; A, A2/4/63; +, A2[23[57; II, rcm~/35; O, rmi/47; Q, PR8/34; D, CAM/46; A, SW/30; V, AI/WA/ 56;, WSE/33; V, NWS/33; [], LEE/ ~ Time (rain.) Fig. 2. Inactivation of RI5 + (m) and RI5- (0) neuraminidases at 55.

8 392 c.k.j. PANIKER Heat stability of viral neuraminidases Among the type A strains, the A2 strains had the most stable enzymes which were unaffected by heating at 45 for I hr (Fig. I). NWS and ws~ had the most labile enzymes. The other strains tested fell into the following descending order of heat stability, which was unrelated to their antigenic cross-reactivity or their times of isolation: MEL, FM I, PR8, CAM, SW and A I/WA/56. The two variants of R 15 strain (type A2) showed different degrees of heat stability (Fig. 2). At 55 R 15 + enzyme retained over half its activity after 2 hr while R 15-- enzyme was almost totally inactivated in I hr. Of the two type B strains tested, B/TAI/62 had a very stable enzyme, unaffected by 45 for 2 hr, while the enzyme of LEE was very labile, being completely destroyed in IO min. The heat-labile enzymes (wse, NWS) not only lost their enzymic activity rapidly but also their antigenicity. It was essential to use freshly prepared suspensions of these viruses in order to prepare anti-neuraminidase sera. DISCUSSION The neuraminidases of influenza viruses differed antigenically from one another, showing varying degrees of cross-reaction. The neuraminidases of type A and B strains were antigenically unrelated. The enzymes of type A strains fell into three distinct antigenic groups (I) sw, (2) Ao-AI and (3) A2. The members of the Ao-AI group were all inter-related and no clear cut distinction could be made between Ao and A I strains on the basis of the antigenicity of their enzymes. The enzymes of type A2 strains did not cross-react with those of sw or Ao-A I. The neuraminidases of the four type B strains tested were all inter-related. Within each group (Ao-A I, A2, and B), the members showed evidence of antigenic drift. There was no cross-reaction between the haemagglutinins of influenza types A and B, but with type A strains there was some cross-reaction between the members of the different subgroups. In spite of this, it was possible to distinguish the subgroups sw, Ao, A I and A2, different strains in the last three subgroups showing antigenic drift. The major shift in the HA antigen was between the A2 and the earlier type A strains of subgroups Ao and A I. The HA and neuraminidase are two separate antigens which stimulate the production of two different antibodies (Webster & Laver, under publication). It is therefore not surprising that the pattern of cross-reaction between different strains is not the same in respect of the two antigens. The recognition of antigenic drift in neuraminidases offers a new parameter in the serology of influenza, both antigens in the envelope show antigenic drift whereas the internal nucleoprotein antigens are antigenically much more stable. The occurrence of antigenic drift in the neuraminidase suggests that antigenic changes in the enzyme confer some survival advantage on the virus. Though several roles have been suggested for the viral enzyme (Rafelson, Wilson & Schneir, I962), none has as yet been conclusively proved. Recent studies in tissue culture (Seto & Rott, I966) and in pieces of chorioallantois on shell (Fazekas de St Groth, Withell & Lafferty, 1958) indicate that the enzyme might be concerned with the release of progeny virus from infected cells. In infected human beings, anti-neuraminidase antibody may,

9 Antigenic drift in influenza virus enzymes 393 by analogy, hinder the dissemination of the virus and thus limit its spread in the community. It has been reported that the neuraminidases of early A o strains were heat labile and those of A2 strains very heat stable (Seto, Hickey & Rasmussen, I959; Rasmussen, 1964). Tests made with the same strains as those used for the antigenic analysis showed that different strains varied greatly in the heat stability of their enzymes, but that, unlike the antigenicity of the neuraminidase, the heat stability had no direct relationship to the time of isolation of the virus. The enzymes of strains NWS and WSE were heat-labile in both their enzyme activity and their ability to induce anti-enzyme antibody. The lack of relationship between A2 neuraminidase and those of earlier A strains is important when considering the origin of the pandemic strains. The regular antigenic drift in both the haemagglutinin and neuraminidase antigens in Ao and A I strains is almost certainly due to mutation and selection of pre-existing strains, but it is very unlikely that the Az strains, which appeared suddenly with neuraminidase and haemagglutinin antigenically unrelated to those of the A I strains of 1956, evolved in this manner. The present findings support the idea (Andrewes, 1959) that A2 influenza virus originated from an animal reservoir. This work was carried out while on deputation from the Government of Kerala, India, under the Colombo Plan. I am grateful to Dr R. G. Webster for his help and guidance and to Professor F. J. Fenner for his interest in the work. REFERENCES ADA, G. L., LIND,'P. E. ~. LAVER, E. G. (1963). An immunological study of avian, viral and bacterial neuraminidase based on specific inhibition of enzyme by antibody. J. gen. MicrobioL 32, 225. AMINOFF, D. (I96I). Methods for the quantitative estimation of N-acetyl neuraminic acid and their application to hydrolysates of sialomucoids. Biochem. J. 81, 384. ANDERSON, S. G. & BURNET, F. M. (1947). Sporadic and minor epidemic incidence of influenza A in Victoria, I. Phase behaviour of influenza A strains in relation to epidemic characteristics. Aust. J. exp. Biol. reed. Sci. 25, 235. ANDREWES, C. H. (I959). Asian influenza: a challenge to epidemiology. In Perspectives in Virology, p (Vol. 1, edited by M. Pollard.) New York: John Wiley and Sons. BURNET, F. M. (1935). Influenza virus isolated from an Australian epidemic. Aust. J. Med. 2, 65I. B~RNET, F. M. (I95I). A genetic approach to variation in influenza viruses. I. The characters of three substrains of influenza virus A (WS). J. gen. Microbiol. 5, 46. BURNET, F. M. (1955). Principles of Animal Virology, p New York: Academic Press Inc. CHOPPIN, P. W. TAMM, I. (I959). TWO kinds of particles with contrasting properties in influenza A virus strains from the 1957 pandemic. Virology 8, 539. DRZENIEK, R., SETO, J. T. dr. ROTT, R. (1966). Characterization of neuraminidases from myxoviruses. Biochim. biophys..4cta x28, 547. FAZEKAS DE ST GROTH, S., GRAHAM, D. i. & JACK, I. (I958). The serology of mumps infection. I. A new source of antigen and a simplified complement-fixation test. J. lab. clin. ivied. 5I, 883. FAZEKAS DE ST GROTH, S., WITHELL, J. & LAFFERTY, K. J. (1958). All improved assay method neutralizing antibodies against influenza viruses. J. Hyg., Cam& 56, 4x5. FRANCIS, T. (194o). A new type of virus from epidemic influenza. Science, N.Y. 92, 4o5. FRANCIS, T. & MAASSAB, H. F. (1965). Influenza viruses. In Viral and Rickettsial Infections of Man, 4th ed., p Edited by F. L. Horsfall and I. Tamm. Philadelphia: J. B. Lippincott Co. FRANCIS, T. & MAGILL, T. P. (I935). Immunological studies with the virus of influenza. J. exp. Biol. 62, 505.

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