A new subtype of avian influenzavirus: antigenic characteristics of envelope antigens

Bull. Org. mond. Sante6 1972, 47, 503-506 Bull. Wid Hlth Org.J A new subtype of avian influenzavirus: antigenic characteristics of envelope antigens BELA TUMOVA,' VALENTINA ISAtENKO,2 & B. C. EASTERDAY3 The haemagglutination and neuraminidase antigens of three influenza A isolates from ducks in the Ukraine were compared with those of a collection of reference strains of influenza A virus. Duck/Ukraine/1/60 virus contained haemagglutinin related to that of duck/England/56 while its neuraminidase was related to that of turkeyl Wisconsin/68 virus and the human A/Hong Kong/l/68 virus. Duck/Ukraine/2/60 and duck/Ukraine/l/63 were themselves closely related. They contained haemagglutinin antigens unrelated to the six haemagglutinin subtypes previously describedfor avian influenzaviruses and it is suggest- ed that they should be classified as belonging to haemagglutinin subtype Hav7. The neura- minidase antigens of these isolates were antigenically related to those of a number of other avian influenza viruses isolated in England, Canada, and Italy and to that of Alequinef Miami/63 virus. The first isolations of type A influenzaviruses from ducks were reported from Czechoslovakia and Eng- land (Andrewes & Worthington, 1959; Koppel et al., 1956). Subsequently avian influenzaviruses were isolated in South Africa, northern Scotland, Italy, Canada, and in the USA (Wells, 1963; Lang et al., 1963; Roberts, 1964; Pereira et al., 1966, 1967). Antigenic characterization of these strains has reveal- ed that some of them contain surface antigens re- lated to those of type A influenzaviruses isolated from pigs, horses, and man (Pereira et al., 1967; Tumova & Pereira, 1968; Schild & Newman, 1969; Schild et al., 1969; Tumova & Easterday, 1969). In 1960-63, nine myxoviruses were isolated in the USSR from cases of duck sinusitis; the disease was widespread among ducklings in the southern regions of the Ukraine (Prokof'eva & Tsimokh, 1966). Of these viruses, two were originally desig- nated " Jalta " and " Borki " and were isolated in 1960. The previously unidentified strain " BVI " was isolated in the same area in 1963. In this paper these strains are designated duck/Ukraine/1/60, duck/Ukraine/2/60, and duck/Ukraine/1/63, respec- I Czechoslovak Influenza Centre, Institute of Hygiene and Epidemiology, Prague, Czechoslovakia. WHO Regional Influenza Centre, Ivanovskij Institute of Virology, Moscow, USSR. 3Department of Veterinary Science, University of Wisconsin, Madison, Wisconsin, USA. tively. The antigenic relationships of these strains to influenzaviruses isolated from several avian and mammalian species including human beings have been studied. MATERIALS AND METHODS Viruses The histories and characteristics of the strains employed in this study have been described pre- viously (Tumova & Easterday, 1969; Easterday & Tumova, 1970). Sera Rat sera. 3-ml volumes of allantoic fluid contain- ing 103 EID50 of virus were injected intraperitoneally (4 times at 3- or 4-day intervals). A fifth injection was given 6 weeks after the first. The antigen for the second to fifth injections had been purified by adsorption on chicken erythrocytes and elution into saline. The rats were bled 14 days after the fifth injection. Antisera. Guineapig strain-specific V antisera were prepared by the technique described by Lief & Henle (1959). Ferret sera. Ferrets were infected intranasally with 0.2-ml volumes of allantoic fluid containing 103 EID50 of virus and reinfected in the same manner 4 weeks after the primary infection. Serum specimens 2924 - 503 BELA TUMOVA, VALENTINA ISAtENKO & B. C. EASTERDAY were taken before the infection, after primary in- fection, before reinfection, and 16 days after rein- fection. Sera were inactivated at 56°C for 45 min for all serological tests and were treated with receptor- destroying enzyme before use in haemagglutination inhibition (HI) tests (WHO Expert Committee on Influenza, 1959). Serological methods HI tests were performed by microtitration in Linbro plastic trays using 0.025-ml volumes. Mix- tures of serum and 4 haemagglutinin units of virus were incubated at room temperature for 1 h before adding erythrocytes (0.7% suspension). Complement fixation (CF) tests were performed as described by Pereira et al. (1964). Neuraminidase inhibition (NI) tests were performed by the method described by Webster & Pereira (1968). The results of serological tests were expressed as reciprocals of the serum dilution giving 50% inhi- bition. RESULTS The viruses under study share the type-specific ribonucleoprotein antigen, common to all type A influenzaviruses. The relationships of the surface antigens, haemagglutinin, and neuraminidase were determined by means of the HI test, the strain- specific CF test, and the NI test. The results are summarized in Tables 1 and 2. It is evident from the results of the HI tests with hyperimmune rat sera that duck/Ukraine/l/60 is antigenically related to duck/England/56 by its haemagglutinin antigen and to turkey/Wisconsin/68 by its neuraminidase antigen (Table 1). There were no cross-reactions in HI or NI tests with other avian, equine, porcine, and human type A influenza- viruses. The duck/Ukraine/l/60 virus was found to be antigenically distinct from the duck/Ukraine 2/60 and duck/Ukraine 1/63 viruses. The duck/Ukraine/2/60 and 1/63 viruses are mutu- ally related and are distinct from previously isolated avian influenzaviruses with respect to their haemag- glutinin content (Table 2). However, both are re- lated by their neuraminidase antigens to other avian influenzavirus strains, isolated at different times in different countries, belonging to different avian subtypes with regard to their haemagglutinin. No relationship was demonstrated to porcine (Hswl N1), equine (HeqlNeql), and human (HONI and HINI) influenzaviruses. The relationship of the envelope antigens of duck/Ukraine/2/60 and duck/Ukraine/1/63 with those of equine/Miami/63 and equine/Detroit/63, and the human A/Hong Kong/1/68 viruses have been report- ed previously (Tumova & Easterday, 1969) and were confirmed in the present study by HI and NI tests using hyperimmune rat sera. Also, experiments with sera from ferrets experimentally infected with duck/ Ukraine/2/60 and duck/Ukraine/1/63 indicated a rise in antibody to the Heq2 haemagglutinin and to that of A/Hong/Kong/1/68 virus suggesting an antigenic re- lationship between the envelope antigens of these viruses and those of the duck/Ukraine isolates. DISCUSSION Until now, avian influenzaviruses have been divided into 6 subtypes based on the results of the HI, CF, and virus neutralization tests (Pereira et al., 1965, 1966, 1967). The antigenic character of the haemag- glutinins of avian influenzaviruses has been used as the basis for their separation into antigenic groups and the present studies suggest that duck/Ukraine/l/ 60 has the same haemagglutinin subtype (Hav3) as duck/England/56. The duck/Ukraine/l/63 and duck/Ukraine/2/60 viruses, antigenically similar in both envelope anti- gens, contain haemagglutinin that is different from any of the 6 proposed avian haemagglutinin subtypes and forms a new subtype Hav7; their neuraminidase antigens are similar to those of some strains with Hav2 or Hav6 haemagglutinin. There is, however, no strict dividing line between subtypes. Antigenic overlapping occasionally occurs between strains when a virus antigenically " bridges " two different subtypes because of a similar haemagglutinin or neurami- nidase; " antigenic bridges " also occur between influenzaviruses of different animal species, as is demonstrated by the relationship between duck/ Ukraine/2/60,1/63, equine/Miami/63, A/Hong Kong/ 68 and other viruses (Webster & Pereira, 1968; Tumova & Pereira, 1968; Schild & Newman, 1969; Tumova' & Easterday, 1969). Antigenic characteri- zation of new influenzavirus isolates becomes even more complicated when both of the known envelope antigens are taken into consideration. The present studies have shown that duck/Ukraine/ 1/60 is not related to duck/Ukraine/2/60 and duck/ Ukraine/1/63 even though they were isolated from the same species in the same area. Similar obser- vations of the isolation of antigenically different influenza A viruses from the same avian or mammal- 504 A NEW SUBTYPE OF AVIAN INFLUENZAVIRUS: ANTIGENIC CHARACTERISTICS OF ENVELOPE ANTIGENS 505 Table 1. Haemagglutinin and neuraminidase antigens of duck/Ukraine/i/60 virus Antigens Sera a duck/Ukraine/i /60 duck/England/56 turkey/Wisconsin/68 HI NI HI NI HI NI duck/Ukraine/i /60 160 90 320 c b 10 duck/England/i /56 (Hav3 Navl) 20 b 640 c b b turkey/Wisconsin/68 (Hav5N ?) b 60 b c 240 40 a Hyperimmune rat sera. b Results were negative in the initial serum dilution (1: 10). c Not tested. Table 2. Antigenic relationships between envelope antigens of avian influenzaviruses Homolo- Antigens (immunestrain gous duck/Ukraine/i /60 duck/Ukraine/2/60 duck/Ukraine/i /63(immune sera) HI _____________________ ____________ titre HIa NIa HIa CFb NIa Hla CFb Nla turkey/England/63 (Havl Nav3) 640 duck/Canada/53 (Hav2 N?) 640 chick/Germany/N/49 (Hav2 Neq2) 1 280 quail/Italy/1117/65 (Hav2 Neq2) 2 560 15 >100 >100 quail/Italy/544/66 (Hav2 Neq2) 1 280 40 >100 5 >100 pheasant/italy/647/66 (Hav2 N?) 7 280 40 50 duck/England/56 (Hav3 Navi) 640 20 duck/Ukraine/i /60 160 160 90 duck/Czechoslovakia/56 (Hav4 Navi) 120 320 >100 >100 chicken/Scotland/59 (Hav5 N1) 320 turkey/Ontario/621 3/66 (Hav5 N1) 320 turkey/Wisconsin/68 (Hav5 N ?) 640 60 turkey/Canada/63 (Hav6 Neq2) 240 40 80 10 100 turkey/Massachusetts/65 (Hav6 N2) 640 turkey/Wisconsin/66 (Hav6 N2) 2 560 turkey/England/66 (Hav6 N?) 1 280 duck/Ukraine/2/60 duck/Ukraine/i /63 80 640 80 80 160 80 350 80 20 2 230 310 640 40 1 000 * Blank spaces denote negative results in the initial 1: 10 serum dilution. a Hyperimmmune rat sera were used for HI and NI tests. b CF tests were carried out using V antigens and V antisera. 506 BELA TUMOVA, VALENTINA ISAtENKO & B. C. EASTERDAY ian species in the same locality or even the same epidemic have been made previously (Pereira et al., 1967; Smithies et al., 1969). Special attention is directed to the fact that the duck/Ukraine/2/60 virus was circulating in an avian population in the Ukraine 3 years prior to the iso- lation of the equine/Miami/63 virus from horses in the USA and 8 years prior to the isolation of the A/Hong Kong/68 virus from human beings of Hong Kong. RESUME UN NOUVEAU SOUS-TYPE DE VIRUS DE LA GRIPPE AVIAIRE: CARACTE-RISTIQUES ANTIGENIQUES DES ANTIGENES D'ENVELOPPE On a compare l'hemagglutinine et la neuraniinidase de trois souches de virus grippaux A isol6es chez des canards en Ukraine avec celles de diverses souches de reference de virus grippaux A. Le virus duck/Ukraine/1/60 renferme une hemagglutinine apparentee A I'hemagglu- tinine du virus duck/England/56, alors que sa neuramini- dase est antigeniquement proche de celles des virus turkey/Wisconsin/68 et A/Hong Kong/1/68. I1 existe une etroite parente antigenique entre les virus duck/Ukraine/ 2/60 et duck/Ukraine/l/63. Leurs hemagglutinines ne pr6sentent aucun lien antigenique avec les six sous-types d'hemagglutinines de virus aviaires precedemment decrits et on propose de les classer dans un nouveau sous-type d'hemagglutinines, le sous-type Hav7. Les neuraminidases de ces virus offrent des affinites antigeniques avec celles d'un certain nombre de virus de la grippe aviaire isoles en Angleterre, au Canada et en Italie, ainsi qu'avec la neuraminidase du virus A/equine/Miami/63. REFERENCES Andrewes, C. H. & Worthington, G. (1959) Bull. Wld Hlth Org., 20, 435-443 Easterday, B. C. & TumovA, B. (1970) Avian influenza. In Hofstad M. J., ed., Diseases of poultry, 6th ed., Ames, Iowa State University Press, pp. 670-700 Koppel, Z. et al. (1956) Veterindrstvi, 6, 26-27 Lang, G. et al. (1963) Avian Dis., 5, 493-504 Lief, F. S. & Henle, E. (1959) Bull. Wld Hlth Org., 20, 411-420 Olesiuk, G. W. et al. (1967) Avian Dis., 11, 203-208 Pereira, H. G. et al. (1964) Bull. Wld Hlth Org., 31, 129-132 Pereira, H. G. (1965) Bull. Wld Hlth Org., 32, 855-860 Pereira, H. G. et al. (1966) Bull. Wld HIth Org., 35, 799-802 Pereira H. G. et al. (1967) Bull. Wld Hlth Org., 37, 553-558 Pereira, H. G. et al. (1967) Nature (Lond.), 215, 982-983 Prokof'eva, U. T. et al. (1963) Veterinarija, 40, No. 10, pp. 33-35 Prokof'eva, M. T. & Tsimokh, P. F. (1966) [Virus influenza of ducks]. In: [Poultry diseases], Moscow, Kolos, pp. 95-106 (in Russian) Roberts, D. G. (1964) Vet. Rec., 75, 470473 Schild, G. C. et al. (1969) Nature (Lond.), 222, 1299 Schild, G. C. & Newman, R. W. (1969) Bull. Wld Hlth Org., 41, 437-445 Smirnova, G. A. & Stakhanova, V. M. (1964) Veterinarija, 41, No. 4, pp. 7-12 Smithies, L. K. et al. (1969) Avian Dis., 13, 606-610 Tsimokh, P. F. (1961) Veterinarija, 38, No. 12, pp. 63-65 Tumova, B. & Pereira, H. G. (1968) Bull. Wld Hlth Org., 38, 415420 Tumova, B. & Easterday, B. C. (1969) Bull. Wld Hlth Org., 41, 429435 Webster, R. G. & Pereira, H. G. (1968) J. gen. Virol., 3, 201-208 Wells, R. J. H. (1963) Vet. Rec., 75, 783 WHO Expert Committee on Respiratory Virus Diseases (1959) Wld Hlth Org. techn. Rep. Ser., No. 170