Properties of the Hong Kong influenza virus

Bull. Org. mond. Sante 1969, 41, 419-424 Bull. Wld Hlth Org. Properties of the Hong Kong Influenza Virus 2. Antigenic Relationship of the Hong Kong Virus Haemagglutinin to that of Other Human Influenza A Viruses WALTER R. DOWDLE,1 MARION T. COLEMAN,1 ELMER C. HALL 2 & VIOLETA KNEZ 3 The magnitude of antigenic change of the Hong Kong strains from the earlier influenza A2 strains was compared with previous antigenic changes among the type A viruses. Re- presentative type A strains recovered from man during the years 1933 through 1969 were examined by reciprocal haemagglutination-inhibition, neutralization and neuraminidase- inhibition tests, using antisera produced in chickens. Phenograms or "family trees " were constructed, using numerical taxonomic methods based on cluster analysis of similarity coefficients. By the use of numerical taxonomic methods the inequality of the antigenic relationships among presently recognized classes becomes evident. Antigenic dissimilarities between AO and Al strains seen in phenograms constructedfrom reciprocal haemagglutina- tion-inhibition and neutralization tests were of lower magnitude than dissimilarities between Al and A2 strains and should not be given equal emphasis. The degree of antigenic dis- similarity between the Hong Kong-like strains and the early A2 strains was even less than that between AO and Al. The authors stress the need for a new system for identifying and classifying human influenza A strains. During the early days of the Hong Kong influenza epidemic and the months following, our laboratory, which serves as the WHO International Influenza Center for the Americas, was under considerable pressure from epidemiologists, public health labora- tories, and the press to designate the Hong Kong virus as A3. It was argued that the epidemic was unlike any experienced since the emergence of the A2 subtype in 1957, that the virus was antigenically only distantly related to A2, that the periodicity of 11 years was almost the same as that between Al and A2, and, more important, that there was a real need for a name which would distinguish this virus from all previous strains. 1 WHO International Influenza Center for the Americas and the Respiratory Virology Unit, National Communicable Disease Center, Health Services and Mental Health Admin- istration, Public Health Service, US Department of Health, Education, and Welfare, Atlanta, Ga., USA. ' Statistical Activities, Laboratory Division, National Communicable Disease Center, Health Services and Mental Health Administration, US Department of Health, Educa- tion, and Welfare, Atlanta, Ga., USA. ' WHO Fellow, on leave from the Instituto de Virologia, C6rdoba, Argentina. We were, indeed, sympathetic. The arguments clearly illustrated the weaknesses of our present system of identifying influenza viruses. Precise ground rules are lacking. The emergence of the Hong Kong variant forces us to revaluate current concepts of classification and to re-examine earlier antigenic changes within the whole family of human influenza A viruses. ANTIGENIC STUDIES OF INFLUENZA A STRAINS Twenty-seven influenza A strains were selected to represent years and epidemiological events from 1933 to 1968. Antisera were produced in chickens by a single injection of infected allantoic fluid and the animals were bled 10-14 days later. Reciprocal haemagglutination-inhibition (HI) tests (Davenport & Minuse, 1964) were performed in duplicate by the microtechnique and the test results were averaged. Upon casual examination of the results (Table 1) two features are immediately apparent: (1) the antigenic change from Al to A2 was abrupt and discontinuous; and (2) the antigenic changes from 2384 419- W. R. DOWDLE AND OTHERS TA RECIPROCAL HAEMAGGLUTINATION-INHIBITION TE' Antigen Antiserum AntigenWS PR BH Melb. I Hick. Weiss Cam. FM Roma [ FW Eng. I FL) A/WS/33 A/PR/8/34 A/BH/35 A/Melbourne/35 A/Hickcox/40 A/Weiss/43 Al /Cam./46 Al /FM/l /47 Al /Roma/49 Al/FW/l/50 Al/England/1 /51 Al/FLW/l/52 Al /Malaya/302/54 Al /Hawaii/56 Al/Denver/I /57 A2/Japan/305/57 A2/Ann Arbor/2/60 A2/Japan/1 70/62 A2/Taiwan/1 /64 A2/A Ibany/3/65 A2/Ann Arbor/7/67 A2/Tokyo/3/67 A2/Hong Kong/8/68 A2/Aichi/2/68 A2/Cali 'ornia/24/69 A2/Oregon/1 /69 A2/Georgia/25/69 804 80 80 113 28 80 40 10 20 10 10 20 804 113 113 10 10 20 40 320 14 10 80 80 402 226 452 226 160 80 14 56 10 640 10 640 80 20 10 10 56 28 40 226 160 56 509 40 14 14 10 14 40 452 640 2036 905 452 28 113 20 160 20 10 10 40 226 80 160 640 226 34 48 20 160 40 80 40 113 226 380 24 24 24 160 12 10 28 10 160 28 113 269 380 380 80 48 226 24 17 20 80 113 80 640 538 95 320 113 1 522 56 95 it 1(4 1' 8C 4( 2E 21 11 644 66 80 80 a 4 HA units of antigen; chicken red blood cells; chicken antiserum pretreated with RDE. Results expressed as recipr AO (A) to Al and from A2 to the Hong Kong variants were gradual and continuous. Any further comparison of strains by visual inspection of the data in Table 1 is a discouraging task. To obtain a better perspective of the antigenic relationships among strains, a computer was used to construct phenograms, or " family trees ", using numerical taxonomic methods based on cluster analysis of similarity coefficients. This method was first employed with a limited number of influenza B strains by Lee (1968) and later for influenza B and influenza A2 strains by Lee & Tauraso (1968). The results of such analyses of the HI data pres2nted in Table 1 are shown in Fig. 1. By drawing lines across the phenogram at certain points along the ordinate we can divide the strains into a number of antigenic subgroups. These lines are referred to as phenon lines. A line drawn at the zero correlation level in Fig. 1 divides the 27 strains into 2 major groups. The first consists of all strains isolated prior to the 420 ANTIGENIC RELATIONSHIPS OF HUMAN INFLUENZA A HAEMAGGLUTININS JLTS WITH HUMAN INFLUENZA VIRUS TYPE A STRAINS, 1933-69 Antiserum aya ! Hawaii Denver 305 AA/60 170 L TW Alb. AA/67 Tokyo [ HK L Aichi Cal. Oregon Georgia !8 'O .56 j8 38 14 28 50 52 40 28 134 134 10 17 10 160 226 320 160 320 160 160 40 40 14 14 40 14 10 226 254 320 320 320 56 56 40 14 40 28 80 10 320 160 1 018 320 452 80 56 28 10 14 14 80 10 20 80 40 113 804 452 40 40 14 14 10 20 14 40 160 113 452 1 280 2036 226 160 28 14 40 20 160 10 10 40 40 40 320 56 10 metric mean titres of duplicate tests. No entry Indicates a geometric mean titre <1 :10. 1957 Asian influenza epidemic and the second con- sists of all strains isolated since that time. This division is probably the only one on which no one will disagree. Thereafter, the basis for additional subdivision becomes less clear, but strains can be further subdivided by arbitrarily selecting certain levels of correlation. A phenon line drawn at the next major level divides the strains into 3 groups corresponding generally to subtypes AO, Al and A2. A phenon line drawn at the correlation level separat- ing the Hong Kong strains from earlier A2 strains results in a further subdivision of the 27 type A strains into a minimum of 6 or a maximum of 10 groups. Reciprocal neutralization tests with a limited number of strains were performed, utilizing the egg- bit procedure (Fazekas de St. Groth & White, 1958) and the same sera as employed for the HI test. The phenogram (Fig. 2) constructed from the neutraliza- tion results in Table 2 again reveals 2 major 421 28 40 160 160 160 160 640 14 14 80 40 20 80 80 80 28 20 640 320 640 452 640 28 20 56 80 40 14 10 640 254 640 452 1 280 56 28 113 80 56 40 905 320 804 905 280 56 20 56 80 56 905 320 1 280 640 1 280 10 113 56 320 113 160 20 640 320 640 640 804I 422 W. R. DOWDLE AND OTHERS FIG. I PHENOGRAM ILLUSTRATING RELATIONSHIPS OF HUMAN INFLUENZA VIRUS TYPE A STRAINS (1933-69) BY RECIPROCAL HAEMAGGLUTINATION-INHIBITION TESTS a Strains A/WS/33 A/PR/8/ 34 A/Melbourne/35 A/Weiss/43 A/BH/35 A/Hickcox /40 Al/Corn./46 AI/FM/ 1/47 Al/Roma/49 AI/Englond/l/51 AI/Mlolya/302/54 AI/FW/ 1/50 AI/FLW/52 AI/Hawaii/56 Al/ Denver/57 A2/Jopon /305/57 A2/Ann Arbor/2/60 A2/Japon/170/62 A2/Taiwon/l/64 A2/AlbL/3/65 A2/Ann Arbor/7/67 A2/Tokcyo/3/67 A2/Hong Kong/8/68 A2/CaIifornio/24e/69 A2/Aichi /2/68 A2/0regon /1/69 A2/Georgio /25/69 i.0 0.9 0.8 0.7 0.6 0.5 0.4 0.3 0.2 0.1 0.0 -0.1 -0.2 -03 -04 -0.5 -06-0.7 Correlation Coefficient a Data from Table 1. TABLE 2 RECIPROCAL NEUTRALIZATION TEST a RESULTS WITH REPRESENTATIVE HUMAN INFLUENZA VIRUS TYPE A STRAINS, 1934-68 Antiserum Antigen PR Weiss fCam. FM FW T FLW ]Denver 305 [170 Albany Tokyo fAichi A/PR/8/34 2 560 160 40 20 80 20 AIWeissI43 160 1 280 640 160 40 80 AI/Cam./46 10 40 10240 160 320 40 20 Al/FM/1/47 20 640 640 640 40 10 Al/FW/l/50 10 10 20 20 160 20 20 A1/FLW/1/52 20 10 160 40 320 2 560 160 AlIDenverIl/57 40 10 10 10 40 80 1 280 A2/Japan/305/57 -320 640 160 20 40 A2IJapanfl170/62 640 320 80 20 10 A2/Albany/3/65 320 640 2 560 160 40 A2/Tokyo/3/67 10 160 40 320 10 A2IAichi/2168 20 40 80 20 320 G2 10-100 1Dso; chicken antiserum pretreated with RDE. No entry indicates no inhibition at 1 :10 serum dilution. ANTIGENIC RELATIONSHIPS OF HUMAN INFLUENZA A HAEMAGGLUTININS FIG. 2 PHENOGRAM ILLUSTRATING RELATIONSHIPS OF HUMAN INFLUENZA VIRUS TYPE A STRAINS (1934-69) BY RECIPROCAL NEUTRALIZATION TESTS a Strains A/PR/8/34 A/Weiss/43 Al/CamJ46 Al/FM/l/47 Al/FW/l/50 Al/FLW/52 Al/Denver/l/57 A2/Japon/305/57 A2/Japon/170/62 A2/Albany/3/65 A2/Tokyo/3/67 A2/Aichi/2/68 _,_,.,._ 1.0 09 08 0.7 0.6 0.5 04 03 0.2 0.1 QO-01-0.2-0.3-4-0.5-0.6 Correlation Coefficient a Data from Table 2. groups-all viruses before 1957 and all viruses since that time. If antigenic differences among the Hong Kong and earlier A2 viruses deserve recognition, a phenon line can be drawn at the correlation coeffi- cient of 0.65, which separates the viruses into 5 groups-exactly the same number as with the IH phenogram (BH strain was not included in this test). FIG. 3 PHENOGRAM ILLUSTRATING RELATIONSHIPS OF HUMAN INFLUENZA VIRUS TYPE A STRAINS (1934-69) BY RECIPROCAL NEURAMINIDASE-INHIBITION TESTS a Strains A/PR/8/34 A/Weiss/43 Al/FM/1/47 Al/Cami/46 Al/FW/l/50 Al/Denver/l/57 Al/FLW/52 A2/Japon/305/57 A2/Japon/170/62 A2/Albany/3/65 A2/Tokyo/3/67 A2/Aichi/2/68 10 09 08 07 06 05 04 03 02 Ql 00-01 -02-0.3-04--05-06-0.78 Correlation Coefficient a Data from Table 3. A third phenogram constructed from the results (Table 3) of the neuraminidase-inhibition (NI) test (Webster & Pereira, 1968), using the same viruses and the same antisera as employed in the HI and neutralization tests, shows a somewhat different pattern of relationships (Fig. 3). The neuraminidase antigen of the Hong Kong variant, unlike the hae- TABLE 3 RECIPROCAL NEURAMINIDASE-INHIBITION TEST a RESULTS WITH REPRESENTATIVE HUMAN INFLUENZA VIRUS TYPE A STRAINS, 1934-68 Antiserum Antigen n PR |Weiss Cam. FM FW FLW DenverT 305 170 Albany Tokyo Aichi A/PR/8/34 1 660 80 118 69 26 A/Weiss/43 631 525 1 350 270 155 32 59 Al/Cam./46 148 2 460 502 252 252 A1/FM/1/47 24 28 2 520 708 332 60 142 A/FW/1/50 28 525 152 44 A1/FLW/1/52 26 76 35 525 955 302 Al/Denver/I/57 22 252 124 447 A2/Japan/305/57 4 080 8 520 6 030 692 1 660 A2/Japan/170/62 1 420 5 250 9 550 1 420 390 A2/Albany/3/64 2 240 3 170 5 020 1 870 759 A2/Tokyo/3/67 339 302 1 000 2 820 1 590 A2/Aichi/2/68 266 258 708 2 630 944 a Infected allantoic fluid; chicken antiserum. No entry indicates <50 % inhibition with undiluted antiserum. 423 W. R. DOWDLE AND OTHERS magglutinin, is quite similar to that of the other A2 strains. The neuraminidase antigen will be discussed at length in subsequent papers. However, the NI pheno- gram is similar to the others in one respect: the viru- ses are clearly divided into the same 2 major groups. DISCUSSION By using numerical taxonomic methods the in- equality of the antigenic relationships among pre- sently recognized classes becomes evident. Anti- genic dissimilarities between the AO and Al strains seen in phenograms constructed from reciprocal HI and neutralization tests are of lower magnitude than dissimilarities between the Al and A2 strains and should not be given equal emphasis. The degree of antigenic dissimilarity between the Hong Kong-like strains and the early A2 strains is even less than that between AO and Al; hence, the A3 designation cannot be supported on antigenic grounds. Yet there is an undeniable need to differentiate between what are now considered AO strains and Al strains and between Hong Kong-like strains and earlier A2 strains. To do this some new system of identification is required. The World Influenza Centre and the International Influenza Center for the Americas are charged with the responsibility of assigning all incoming influenza strains to some category, and we are acutely aware of the need for a new system. Most of us will probably agree on the major crite- ria for identification of human influenza A viruses: 1. The virus must contain soluble or ribonucleo- protein antigen common to that of type A. 2. The virus must be a bonafide isolate from man. Although certain antigenic relationships among influenza A viruses of man and animals are well recognized, to include all influenza A strains into a single scheme at this point would hopelessly and needlessly confuse the system. 3. Influenza A strains must be subtyped on the basis of envelope-antigen relationships, taking into consideration the relative importance of both the haemagglutinin and the neuraminidase antigens. This proposal comes at a time when the effect of neuraminidase-inhibition antibody on the measure- ment of haemagglutinin antigen is being questioned. Although there is little evidence to suggest that inter- ference of neuraminidase antibody is a major problem in characterizing the haemagglutinins of wild strains, conflicting viewpoints on this subject must be thoroughly resolved. 4. Any subdivision of influenza A strains must be based on equal antigenic classes. We shall resist the temptation here to set forth our own proposals. Rather we suggest that the proposal of any new scheme for identification, the decision regarding additional subdivision and the selection of prototype strains require international collabora- tion. A committee, sponsored by WHO, for in- stance, would be a logical body to perform this function. REFERENCES Davenport, F. M. & Minuse, E. (1964) Influenza viruses. In: American Public Health Association, Diagnostic procedures for viral and rickettsial diseases, 3rd ed., New York, p. 462 Fazekas de St. Groth, S. & White, D. 0. (1958) J. Hyg. (Lond.), 56, 151 Lee, A. M. (1968) Nature (Lond.), 217, 620 Lee, A. M. & Tauraso, N. M. (1968) Bull. Wld Hlth Org., 39, 261 Webster, R. G. & Pereira, H. G. (1968) J. gen. Virol., 3, 201 424