Antigenic variation of equine (Heq2Neq2) influenzavirus

Bull. Org. mond. Sante 1972, 47, 465-469 Bull. Wld Hlth Org. Antigenic variation of equine (Heq2Neq2) influenzavirus H. G. PEREIRA,1 S. TAKIMOTO,2 N. S. PIEGAS 8, & L. A. RIBEIRO DO VALLE2 Influenza equine (Heq2Neq2) strains isolated during the course of epizootics observed in Guanabara (Rio de Janeiro) and Sao Paulo, Brazil, in July-October 1969 were shown to differ antigenically from earlier strains of the same subtype (Afequine/Miami/J/63 (Heq2- Neq2)). The difference could be clearly demonstrated in haemagglutination inhibition tests performed with postinfection horse or ferret sera but not with hyperimmune rooster sera. Antibody responses ofdiseased horses were higher and morefrequent against current isolates than against strain equine/Miami/J/63. Some animals also showed antibody responses to the Hong Kong variant ofhuman influenzavirus A. All the type A influenzaviruses isolated from man, horses, pigs, and birds have a common type-specific ribonucleoprotein antigen and have surface antigens (haemagglutinin and neuraminidase) that undergo variation and allow subtypes and antigenic variants to be distinguished within strains from each of the host species (see review by Pereira, 1969). According to the antigenic specificity of the viral haemagglutinin, human type A influenzaviruses are divided into several subtypes, HONI, HINI, H2N2, and H3N2, there being considerable antigenic varia- tion within each subtype. Equine influenzaviruses are similarly divided into 2 subtypes, equine (Heql- Neql) and equine (Heq2Neq2). The distinction of subtypes and antigenic variants depends, however, on a number of factors, such as the methods of assay, the kind of reagents used, and the choice of strains included in comparative tests. Anti- genically related surface antigens have been observed between subtypes from different hosts (Tumova & Pereira, 1968), and of particular relevance to the present study are the cross-reactions between human and equine strains reported by Coleman et al. (1968) and Kasel & Couch (1969). In the course of equine influenza epizootics ob- 1 Head, Division of Virology, National Institute for Medical Research, Mill Hill, London, England. 'Department of Virology, Instituto Adolfo Lutz, Sao Paulo, Brazil. 3Department of Animal Virology, Instituto Biologico, Sao Paulo, Brazil. ' Head, Department of Virology, Instituto Adolfo Lutz, Sao Paulo, Brazil. served in Brazil from July to October 1969, the virus strains isolated were found to belong to sub- type equine (Heq2Neq2) but to differ significantly from the original A/equine/Miami/1/63 isolate of this subtype. These findings and their implications are discussed in this paper. MATERIALS ANX) METHODS The outbreaks observed took place in racing clubs in Siao Paulo and Rio de Janeiro and in horses belonging to the Sao Paulo police force. The numbers of horses examined were as follows: Sio Paulo racing club, 13; Rio de Janeiro racing club, 6; Sao Paulo police, 13. The animals observed in the racing clubs showed typical signs of equine influenza when first examined in mid-July 1969 whereas those of the police force showed no evidence of infection at that time but developed symptoms about 2 months later. All animals were bled when first examined and were re-bled at different time intervals. Nasal swabs were collected from all but one of the racing horses and from 4 of the police horses which had just recovered from infection. Virus isolation Nasal swabs were submerged in a transport medium consisting of nutrient broth containing 1000 IU of penicillin, 1000 IU of streptomycin, and 5 IU of amphotericin B per ml. Each sample was inoculated into the amniotic cavity of embryonated eggs and incubated at 35°C for 72 hours. After being 2918 - 465- H. G. PEREIRA ET AL. chilled at 4°C overnight the allantoic and amniotic fluids were harvested separately and tested for haemagglutination against a 0.5% suspension of chicken erythrocytes. Haemagglutinin titrations and haemagglutination inhibition tests were performed by the micromethod of Takatsy (1955) using 0.025-ml volumes. Serial 2-fold serum dilutions were tested against 4 haemag- glutinating doses of virus. Serum-virus mixtures were kept for 1 hour at room temperature before the addition of chicken erythrocytes (0.5% suspen- sion). Hyperimmune chicken antisera for strains A/ equine/Prague/1/56 (HeqlNeql) and A/equine/ Miami/1/63 (Heq2Neq2) were obtained from the WHO Influenza Centre for the Americas (Com- municable Diseases Center, Atlanta, Ga. USA). A hyperimmune serum against A/equine/Sao Paulo/ 6/69 (Heq2Neq2) was obtained from a chicken given 3 weekly intramuscular inoculations of 10 ml of infective allantoic fluid with haemag- glutination titre of at least 1/320 and bled 1 week after the last inoculation. Postinfection ferret anti- sera were obtained from ferrets infected intranasally and bled 3 weeks later. All sera were treated with cholera filtrate (Van der Veen & Mulder, 1950) for removal of nonspecific inhibitors. Antigens used for haemagglutination-inhibition were allantoic fluids from eggs infected with strains A/equine/Prague/1/56, A/equine/Miami/1/63 (both received from the WHO Influenza Centre for the Americas), A/equine/Sao Paulo/6/69, and A/Sao Paulo/101/68 (H3N2). The latter is a human strain indistinguishable from A/Hong Kong/1/68. As this last strain was highly sensitive to nonspecific serum inhibitors when first isolated, it was serially propa- gated in the presence of normal horse serum until an inhibitor-insensitive variant was obtained for use in the tests reported here. RESULTS Virus isolation was positive for 13 of the 22 horses tested. Isolation rates were high in both racing clubs where samples were collected when the ani- mals showed typical symptoms of infection but no isolates were obtained from the four police horses tested, which had largely recovered at the time of sampling. All the isolates were found to belong to the equine (Heq2Neq2) subtype and behaved identi- cally in haemagglutination inhibition tests against chicken antisera to equine/Prague/1/56, equine/ Miami/1/63, and equine/Sao Paulo/6/69. However, reciprocal tests between a representative isolate of the present outbreak (A/equine/Sao Paulo/6/69) and the original equine/Prague/56 and equine/Miami/63 (Table 1) revealed a significant antigenic drift within the equine (Heq2Neq2) strains, neither of which cross-reacted with equine (HeqlNeql). This drift is clearly indicated by the two-way difference between homologous and heterologous haemagglutination inhibition titres given by postinfection but not by hyperimmune sera. Evidence of an antigenic drift is also indicated by the antibody responses of naturally infected horses: Table 2 shows representative HI antibody responses in horses from the three different stables. These results are summarized in Table 3 for all the horses examined serologically. Whereas all but 1 of the horses showed significant antibody rises to the current isolate A/equine/Sao Paulo/6/69, only 21 of 32 horses responded to A/equine/Miami/l/63, the latter responses being generally lower. Antibody rises to A/equine/Prague/56 were de- Table 1. Comparison haemagglutination inhibition titres of equine influenza strains a Hyperimmune chicken sera Postinfection ferret sera Convalescent Strains horse serum equine/ equine/ equine/ equine/ equine/ equine/ equine/ Prague/56 Miami/63 Sao Paulo/69 Prague/56 Miami/63 Sao Paulo/69 Sao Paulo/69 equine/Prague/56 160 <10 <10 320 <10 <10 <10 equine/Miami/63 <10 320 160 <10 640 30 <10 equine/S.P./69 <10 320 160 <10 80 1 920 80 a The figures represent the reciprocals of the serum dilutions corresponding to the 50% HI endpoint. 466 ANTIGENIC VARIATION OF EQUINE VIRUSES 467 Table 2. Haemagglutination inhibition titres of representative horse sera a Antigens Horse Age Sex Serum DtHorse samples ate A/equine/ A/equine/ A/equine/ A/SaoPaulo/ Prague/i /56 Miami/i /63 Sio Paulo/6/69 101 /68 b Racing horses from Rio de Janeiro GB1 3 M 1st 20 July 1969 <10 <10 <10 <10 2nd 3 August 1969 80 80 1 280 30 GB2 6 F 1st 20 July 1969 <10 <10 <10 <10 2nd 3 August 1969 <10 20 <320 <10 GB3 4 F 1st 20 July 1969 <10 <10 <10 <10 2nd 3 August 1969 40 80 >1 280 30 GB4 6 F 1st 20 July 1969 <10 <10 <10 <10 2nd 3 August 1969 <10 640 >1 280 120 Racing horses from Sao Paulo SPi 5 F 1st 16 July 1969 <10 <10 <10 <10 2nd 4 August 1969 <10 <10 80 15 3rd 26 August 1969 <10 <10 40 <10 SP2 3 M 1st 16 July 1969 <10 <10 <10 <10 2nd 4 August 1969 20 30 120 40 3rd 20 August 1969 <10 10 80 20 SP3 4 M 1st 16 July 1969 <10 <10 <10 <10 2nd 4 August 1969 <10 <10 80 <10 3rd 20 August 1969 <10 <10 60 <10 SP4 2 F 1st 16 July 1969 <10 <10 <10 <10 2nd 4 August 1969 <10 <10 80 <10 3rd 20 August 1969 <10 <10 60 <10 Police horses from S3o Paulo P11 14 M 1st 23 July 1969 10 <10 <10 <10 2nd 10 October 1969 10 <10 120 <10 3rd 24 October 1969 10 <10 30 <10 P17 8 M 1st 23 July 1969 <10 <10 <10 <10 2nd 10 October 1969 <10 40 <960 120 3rd 24 October 1969 20 40 320 30 P21 3 M 1st 25 July 1969 60 <10 <10 <10 2nd IO October 1969 60 <10 100 <10 3rd 24 October 1969 60 <10 120 <10 P27 14 M 1st 25 July 1969 20 <10 40 10 2nd 10 October 1969 40 30 80 40 3rd 24 October 1969 40 30 60 30 a Figures represent reciprocal of serum dilutions corresponding to the 50% HI endpoint. b Antigenically indistinguishable from A/Hong Kong/i /68 virus. Table 3. Number of horses showing significant HI antibody rises against different virus strains Antigens Locality A/equine/ A/equine/ A/equine/ A/Sio Paulo/ No. of paired sera Prague/i /56 Miami/i /63 Sao Paulo/6/69 101/68 a Sao Paulo racing club 13 1 6 13 3 Rio de Janeiro racing club 6 2 6 6 5 Sao Paulo police force 13 0 9 12 8 total 32 3 21 31 16 a Antigenically identical to A/Hong Kong/i /68 virus. 3 H. G. PEREIRA ET AL. tected in a few of the animals. This finding is diffi- cult to interpret without information on the pre- vious history of infection of the populations under study. Another finding difficult to explain is the higher level of antibody response shown by horses in Rio de Janeiro compared with those in Sao Paulo. The antibody responses shown by a number of the horses against the human strain A/Sao Paulo/ 101/68 (antigenically identical to A/Hong Kong/1/68 virus) are in agreement with previous findings of an antigenic relationship between the Hong Kong variant and equine (Heq2Neq2) virus. DISCUSSION The present results demonstrate that antigenic variation occurs within the equine (Heq2Neq2) influenza A subtype. This observation brings the equine strains into line with those belonging to sub- types of other hosts. Failure to detect antigenic varia- tion in previous studies of equine (Heq2Neq2) strains may have resulted from the common use of hyper- immune rather than postinfection antisera for the identification of virus isolates. It may be mentioned, however, that antigenic variation of the magnitude observed in the present study has not been detected among the equine (Heq2Neq2) strains studied so far at the World Influenza Centre, where postinfectious ferret sera are routinely used for virus identification. A feature of the present study that has practical implications is that the antibody responses shown by the infected horses were higher and more frequent against the current isolate A/equine/Sao Paulo/6/69 than against the earlier A/equine/Miami/1/63 strain. If only the latter isolate had been used the antibody rises would not have been detected in a considerable proportion of infected animals. The possibility that the differences in antibody titres may have resulted from variations in the avidity for antibody rather than in the antigenic structure is unlikely as both strains reacted equally with the hyperimmune sera and showed a two-way antigenic difference in reciprocal tests with ferret sera. Our results suggest that the antigenic variation observed in the present study occurred at sites distinct from those responsible for the cross-reac- tions previously reported between equine (Heq2- Neq2) strains and the human A/Hong Kong/68 virus. No explanation can be offered of the difference in antibody rises observed in animals from Rio de Janeiro and Sao Paulo or of the low levels and occa- sional rises in antibodies to the subtype equine (HeqlNeql). These findings suggest, however, that some of the animals may have been previously exposed to influenza. ACKNOWLEDGEMENTS The authors acknowledge the valuable help of Dr F. Cavallari and Mr 0. Lenci of the Jockey Club of Sao Paulo, Dr 0. G. Pereira of the Jockey Club of Brazil, Rio de Janeiro, Dr Laerte S. Traldi of the Police Force of Sao Paulo, and Mr V. Zaguetti of the Virology Department, Instituto Adolpho Lutz, Sao Paulo. One of the authors (H. G. P.) was supported by the Foundation for the Support of Scientific Research of the State of So Paulo. RI_SUMI VARIATION ANTIGENIQUE DU VIRUS GRIPPAL EQUINE (HEQ2NEQ2) Des souches de virus equine (Heq2Neq2) ont ete isol6es chez une forte proportion des chevaux appartenant a deux 6curiesdeRiodeJaneiroetdeSao Paulo, au Bresil, aucours d'epizooties en juin-octobre 1969. Des tentatives d'isole- ment de virus, pratiquees plus tardivement sur un troi- si6me groupe de chevaux, a Sao Paulo, n'ont donn6 aucun resultat. Les souches isol6es differaient antig6niquement des souches du meme sous-type (souches A/equine/Miami/ 1/63) identifi6es ant6rieurement. La difference est nette- ment apparue lors des 6preuves d'inhibition de I'h6mag- glutination effectues avec des antiserums de cheval ou de furet; elle ne s'est pas marqu6e avec des s6rums hyper- immuns de poulet. Les serums des chevaux atteints par 468 ANTIGENIC VARIATION OF EQUINE VIRUSES 469 l'epizootie de grippe etaient plus fr6quemment positifs et presentaient des titres d'anticorps plus elev6s en presence du virus A/equine (1969) qu'en presence du virus A/equine/Miami/1/63. Certains des s6rums positifs pour la souche A/equine reagissaient egalement en presence du virus A/Hong Kong/68 de la grippe humaine, confirmant ainsi la parent6 antig6nique entre les hemagglutinines de ces deux virus. Ces observations d6montrent i'existence de variations antig6niques au sein du sous-type A/equine consid6re autrefois comme un groupe viral homogene. REFERENCES Coleman, M. T. et al. (1968) Lancet, 2, 1348 Kasel, J. A. & Couch, R. B. (1969) Bull. Wld Hlth Org., 41, 447 Pereira, H. G. (1969) Progr. med. Virol., 11, 46 Takatsy, G. (1965) Acta microbiol. Acad. Sci. hung., 3, 191 Tumova, B. & Pereira, H. G. (1968) Bull. Wld Hlth Org., 38, 415 Van der Veen, J. & Mulder, J. 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