Antigenic and amino acid sequence analyses of influenza viruses of the H1N1 subtype isolated between 1982 and 1984

Bulletin ofthe WorldHealth Organization, 63(2): 273-277 (1985) ( World Health Organization 1985 Antigenic and amino acid sequence analyses of influenza viruses of the HINI subtype isolated between 1982 and 1984 R. S. DANIELS,' A. R. DOUGLAS,' J. J. SKEHEL,' & D. C. WILEY2 Since the reintroduction of influenza viruses of the HINI subtype into the human population in 1977, antigenically distinguishable viruses have been isolated each yearfrom outbreaks of respiratory disease in young people. In this paper the antigenic properties of viruses representative of isolates made in 1982, 1983, and 1984 are compared, and the amino acid sequences of their haemagglutinins are presented. The results provide a complete description ofthe 1982 and 1983 HINI influenza virus haemagglutinins and allow correlation of specific amino acid sequence differences with antigenic differences. Influenza viruses of the HIN I subtype have caused epidemics of disease in two periods of this century -from about 1918 until 1957 and from 1977 until the present. The reintroduction, in 1977, of a virus similar to the 1950 isolate A/FW/1950 (1-3) marked the first recorded cocirculation in the human population of influenza A viruses of different subtypes. Each year since then, viruses of the HINl subtype have been isolated from young people, who were susceptible because they were born after the end of the first H lN 1 period in 1957. Viruses isolated in different years have been shown to differ in antigenic properties. Routinely such antigenic differences are detected in haemagglutination-inhibition tests using postinfection ferret antisera. Information obtained in this way has been complemented recently by similar tests using antihaemagglutinin monoclonal anti- bodies and by analyses of the amino acid sequences of the haemagglutinins of small numbers of represen- tative viruses (4, 5). Here we present the amino acid sequences of the haemagglutinins of four HINI viruses, chosen to represent the antigenic variety of viruses isolated in 1982, 1983, and 1984, and discuss them in terms of the antigenic structure of the haemagglutinin and arjtigenic drift in viruses of the H I N I subtype. Division of Virology, The National Institute for Medical Research, Mill Hill, London NW7 IAA, England. Requests for reprints should be addressed to Dr Skehel. 2 Department of Biochemistry and Molecular Biology, Harvard University, Cambridge, MA 02138, USA. METHODS Haemagglutination-inhibition tests Haemagglutination-inhibition tests (6) were done using postinfection ferret antisera and turkey erythro- cytes. Nucleotide sequence analysis Nucleotide sequences were determined using the dideoxynucleotide chain terminating procedure of Sanger et al. (7). Each 10-al reaction contained 0.05 mol/litre Tris chloride (pH 8.3), 0.012 mol/litre magnesium chloride, 0.02 mol/litre dithiothreitol, 0.0004 mol/litre dATP, dCTP, dGTP, and dTTP, 15 tg of virus RNA, 3 units of human placenta RNase inhibitor,a 5 units of reverse transcriptase, b and 0.00025 mol/litre of either ddATP, ddCTP, ddGTP or ddTTP. After 2 hours at 42 °C, products were analysed on polyacrylamide gels containing 8% acrylamide. Reactions were primed using the fol- lowing 5'-32P-labelled synthetic oligodeoxynucleo- tides, numbered according to the sequence of PR/8/34 cDNA for haemagglutinin (8): 5-AAAGCAGGGGA-1 5; 161-GACACACTCTG-171; 334-ACTCCGAGAATG-345; ' Bethesda Research Laboratories, Science Park, Cambridge, England. " Life Sciences, St Petersburg, FL 33710, USA. 4523 -273 R. S. DANIELS ET AL. 371-CGACTATGAGG-381; 527-GCTATGGCTGACGG-540; 596-AGAAGTCCTTG-606; 810-GAGGCAAATGG-820. RESULTS Antigenic analysis of the haemagglutinins The results of haemagglutination-inhibition tests, given in Table 1, show the antigenic relationships between the HINI viruses of 1982 and 1983 and the prototypes of the subtypes isolated since 1977. They indicate that considerable antigenic change has occurred since 1977-78, but that clear similarities exist between all viruses isolated since 1980. A/Hong Kong/2/82 and A/Chile/l/83 in particular are very similar to A/England/333/80; antisera against A/Dunedin/27/83 inhibit haemagglutination by most of the viruses, although the virus itself reacts poorly as an antigen; and A/Victoria/7/83, which reproducibly induces very specific antisera in ferrets, reacts well with antisera prepared against A/England/333/80, A/Hong Kong/2/82, and A/ Chile/1/83. Of all the viruses received for analysis in the WHO Collaborating Centre for Reference and Research on Influenza, London, in 1982 and 1983, the most freqently isolated and the most widespread were those antigenically similar to A/Hong Kong/ 2/82 and A/Chile/1/83; viruses like A/Dunedin/27/ 83 were isolated least frequently; viruses like A/Victoria/7/83 became equally prevalent with A/Chile/1/83 in the first quarter of 1984. Amino acid sequences of the haemagglutinins The amino acid sequences of the antigenically important HAI polypeptides of the haemagglutinins were deduced from the nucleotide sequences of the genes for haemagglutinin extracted from A/Hong Kong/2/82, A/Chile/1/83, A/Dunedin/27/83, and A/Victoria/7/83, and are shown in Table 2. For comparison, the amino acid sequence of the A/USSR/90/77 haemagglutinin, as reported previously (5, 9, 10), is also shown. The sequence of A/Hong Kong/2/82 haemagglutinin differs from that of A/USSR/90/77 at 12 positions, 11 of which are shared by all four viruses. In addition, A/Chile/l/83 has changes at positions 46, 53, 138 and 225; A/Dunedin/27/83 also has changes at positions 46 and 53 and has four further substitutions at positions 129, 189, 192 and 225; A/Victoria/7/83, in addition to the changes at 46, 53, and 129, also has a change at position 141. Clearly, the amino acid sequences of these four haemagglutinins are very similar and these results allow them to be arranged in order of relatedness: A/Hong Kong/2/82, A/Chile/ 1/83, A/Dunedin/27/83, and A/Victoria/7/83. The basis of this order is simply that A/Chile/1/83, A/Dunedin/27/83 and A/Victoria/7/83 share common differences from A/Hong Kong/2/82 at positions 46 and 53 and A/Dunedin/27/83 and A/Victoria/7/83 both have a substitution at position 129, which the A/Chile/1/83 haemagglutinin lacks. Interestingly in this connection, the haemagglutinins of all four of these viruses have a common substitu- tion at position 134, which distinguishes them from the haemagglutinins of the 1980 isolates A/England/ 333/80 and A/India/6263/80, reported recently (5). DISCUSSION Analysis of the amino acid sequences of the haemagglutinins of variant influenza viruses forms the basis of detailed studies of the antigenicity of the haemagglutinin and of precise surveillance of influenza variation. The results presented here pro- vide complete descriptions of the 1982 and 1983 HINI influenza virus haemagglutinins. They also allow a tentative correlation between specific amino acid substitutions and antigenic differences. For example, in the haemagglutinin of A/Chile/ 1/83, the changes at residues 138 and 225 are distinctive, while in A/Victoria/7/83, the change at 141 may well be responsible for the induction of highly strain-specific antisera by this virus. These possibilities will be assessed in future studies by analysis of the specificity of discriminating monoclonal antibodies. The antigenic significance of particular amino acid substitutions can also be considered in relation to the antigenically important regions of haemagglutinins of both H3 and HI subtypes (12, 13), established by locating antigenically significant amino acid substitutions in the three-dimensional structure of the haemagglutinin of X-31 (H3N2) virus (11). Four anti- genic sites, designated Sa, Sb, Ca, and Cb, have been proposed in the haemagglutinin of the prototype H IN 1 virus A/PR/8/34, and five sites, designated A, B, C, D, and E, in haemagglutinins of the H3 subtype. In relation to these, the haemagglutinin of A/Hong Kong/2/82 virus appears to have no unique antigeni- cally important amino acid substitutions since the single unique change at position 31 is not included in any proposed antigenic area. For A/Chile/ I /83, A/Dunedin/27/83, and A/Victoria/7/83 the shared change at position 53 is in antigenic area C, a site described in the haemagglutinins of H3 but not H1 subtype viruses. The unique change at position 225 in the haemagglutinin of A/Chile/ 1/83 is in site Ca and is a position known to be recognized by antibodies 274 INFLUENZA HINI VIRUSES, 1982-84 0 0 0 0 0 0 000oO 0 N (0 * 0 N 0 V V 0 00 0 0 0 0 01000 (0 0(C0 Nj 0 0 0 0 0 0 010 00CO 00 NMN (0 (0Q E 0 0 0 0 010 0 0N> N(0ED N NICX CD 0000 01' 0 0 0o 0 0 0 0 0 0 0 0 0 N it CO N N 0 N NCN co N m CV - V CV) O ,CW001 0 0 0o O C')CO v v 0 01 0 0 0 0 0 0 O CD V 0 1 0 000 0 0 0 0 00 - N 00N~~~~ 0 N4 00r-cN00 C') 00 N *-40 co.CO w1 I OD r- C 0 C 0 r- C) 00 275 E 0 b- C 0 I._ 0 coN c 0WI' 0 I : u 0 C') co N (0 Cx 0 co C') CD C4 CJLU CU r- OD r- N. m co 0)o cn cn C, C, L.U) Cla -0 c C 40 L-)0 0. 0, U) c - UM (a0 CL .2 c C _ c 0 0P, -C 0 .) m U) C 0 .0 CU - C "n CR DW C0 .'_ C CU U 0 R. S. DANIELS ET AL. U) Un I U)n U) U) U) 0 ._U) C: z a. ° I zz z - U. cc U- c:, cn cn U- 0. U UJ L cn z UJ IL ~~~~~~n w U J z CY) 00 No N) 0 0 C/) le -c c 0)U) o c 5 :: I C" tD) a (V) 00 r- .2 - 0) cc C/)cn D 0.a -z > O z U U) CL z z U) a- I C,,Z z Z c- U. z c 0 c N 0 00 I: e C.) C.- (V) co r-C4 c ca,cx0 C') r- 00 2 Z0 w a- 8> YY e Y z 0 U) cn z 0 a- 0 C-) U) cn cc: z F0 ,-j U) IL G IL a- z a- c- CC C') 00 co _ oX c rI M C4 (1: 0 OD 0: C.,N 0 a, C) - 0) cn cn Rl > > > > U) U) U) U) U) a- z cn ww w w a. C N 0)C4 4) c C, ) ODO fl 0oa >U)O0> Z O0I D° < U 276 CC z U C, z > 20 J a- w _, U C, z Y z I 0 U) Z I >, > 0 0 U) U .r4 n a na) o o a)a) 0 a) C.) C z E 20 ~0 a D 'a 0) -c 0c +,, a) C) .C a) 0* 0 .0 'a H :'C E C) x 0 o 0 a,Cr 0 U U .) 0 C) c a,U a, 0 0E C ., 0 C E c 0 Cm a, EC: 0 Co *C H CO)00 r- 0 U > INFLUENZA HlNl VIRUSES, 1982-84 277 (13). Both this substitution and that at position 138 are close to the sialic acid binding pocket of the haemagglutinin and may be responsible for the differences in affinity for erythrocyte receptors demonstrated by this virus and A/Dunedin/27/83 in haemagglutination tests. The amino acid substitution at residue 129, shared by A/Dunedin/27/83 and A/Victoria/7/83 haemagglutinins, is at a position previously defined to be in antigenic site Sa and clearly could be responsible in part for the antigenic differences between these two viruses and A/Chile/ 1/83 and A/Hong Kong/2/82. Finally, the additional changes at positions 189, 192, and 225 for A/Dunedin/27/83 and at position 141 for A/Victoria/7/83 are also potentially antigenic: 189 and 192 are in site Sb(B); 225, as for A/Chile/1/83 haemagglutinin, is in site Ca; and position 141 is also in Ca(A). ACKNOWLEDGEMENTS We thank David Stevens, Rose Gonsalves, and Gary White for assistance, and Dr M. -Evered, Dr M. Vicente, Dr W. K. Chang, and Dr F. J. Austin of the WHO Collaborating Centres in Australia, Chile, Hong Kong, and New Zealand, for their collaboration. Financial support was received from the Medical Research Council, the World Health Organization, the National Institutes of Health (Al 13654), and the National Science Foundation (PC-771398). RESUME ANALYSE ANTIGENIQUE ET ANALYSE DES SEQUENCES D'ACIDES AMINES DES VIRUS GRIPPAUX DU SOUS-TYPE HIN I ISOLES ENTRE 1982 ET 1984 Depuis 1977, des virus grippaux du sous-type HINI ont ete isoles au cours d'epidemies de maladies respiratoires chez des enfants et de jeunes adultes et il s'est revel qu'ils differaient au point de vue antigenique chaque annee. Le present article expose les analyses antigeniques et les sequencages des acides amines des hemagglutinines de quatre virus, A/Hong Kong/2/82, A/Chile/ 1/83, A/ Dunedin/27/83 et A/Victoria/7/83, representatifs des virus de ce sous-type, expedies pour analyse au centre col- laborateur OMS de references et de recherche pour la grippe, a Londres, depuis l'annee 1982. Les resultats permettent la comparaison detaillee des hemagglutinines et 1'etablissement de correlations entre des substitutions d'acides amines specifiques et des differences antigeniques. Ils sont examines sous I'angle de la localisation des sites de liaison des anticorps dans la structure triditnensionnelle de l'hemagglutinine et en ce qui concerne les differences struc- turales et antigeniques qui interviennent pour determiner la direction de la derive antigenique. REFERENCES 1. SCHOLTISSEK, C. ET AL. Virology, 89: 613-617 (1978). 2. KENDAL, A. P. ET AL. Virology, 89: 632-636 (1978). 3. NAKAJIMA, K. ET AL. 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