Analysis of antigenic variation in equine 2 influenza A viruses

Bulletin of the World Health Organization, 61(1): 153-158 (1983) (© World Health Organization 1983 Analysis of antigenic variation in equine 2 influenza A viruses V. S. HINSHAW, C. W. NAEVE, R. G. WEBSTER,1 A. DOUGLAS,2 J. J. SKEHEL,2 & J. BRYANS3 Influenza outbreaks involving viruses of the H3N8 subtype (equine 2) often occur in vaccinated horses. For this reason, a series of influenza viruses of the H3N8 subtype were examined to determine ifantigenic variation could be detected in isolates during the period 1963-81. Antigenic analyses with post-infection ferret sera and monoclonal antibodies showed that the haemagglutinins of recent isolates were antigenically distinguishable from the prototype A/eq/Miami/1/63 and that antigenically distinguishable groups of equine 2 viruses co-circulate in the horse population. Based on these studies, it is recommended that a recent equine strain, A/equine/Fontainebleu/J/79 or A/equine/Kentucky/1/81, serve as an additional prototype strain for this subtype. Antigenic variation in equine 2 viruses may be ofepidemiological significance, yet the overall conservation of these strains makes it unlikely that vaccine failures can be attributed solely to antigenic changes in these viruses. A sufficiently potent vaccine, containing a current representative of the most prevalent equine 2 strain, may improve the protection afforded by equine vaccines. In 1980-81, severe outbreaks of influenza occurred in horses in Kentucky and California. These outbreaks were associated with influenza viruses of the H3N8 subtype (equine 2), and both vaccinated and unvaccinated animals were affected. Similar problems have been reported in horses in Europe within the last 2 years (1, 5). This raises the possibility that antigenic variation in equine 2 viruses may have occurred. The need to investigate this aspect has been recognized by WHO (21) because of the disease problem in horses and also because the equine 2 strain with an H3 haemagglutinin (related to that of some human influenza viruses) has been postulated to play a role in the genesis of new human strains. Although equine 2 viruses have been circulating in horses for 18 years, there have been few reports of antigenic variation within this subtype. Pereira (11) reported that isolates from horses in South America differed from the prototype A/equine/Miami/1/63 in haemagglutination inhibition tests with post- infection ferret sera. More recent studies on isolates in Japan (7) and Sweden (5) also suggested antigenic variation within this subtype, whereas similar studies on isolates from England during 1979 (1) indicated l St Jude Children's Research Hospital, P.O. Box 318, Memphis, TN 38101, USA. 2 National Institute for Medical Research, Mill Hill, London, England. 3 University of Kentucky, Lexington, KY, USA. that these viruses were not distinguishable from A/equine/Miami/1/63. In view of the current disease problem, the examination of recent equine isolates for evidence of antigenic variation was necessary. Our results indicate that (a) equine 2 viruses do show antigenic variation and (b) antigenic variants co-circulate in the equine population. MATERIALS AND METHODS Viruses Influenza virus isolates were obtained from the repositories of influenza viruses at WHO collaborating laboratories at St Jude Children's Research Hospital, Memphis, TN, USA, and at the National Institute for Medical Research, Mill Hill, London, England. Recent equine 2 virus isolates from horses in Kentucky, California and Georgia were included in the study. These horses experienced severe disease symptoms, including fever, myalgia, profuse nasal discharge, and, in some cases, pneumonia. The California isolate (kindly provided by Dr A. Ardans, University of California, Davis) was recovered from lung tissues obtained at autopsy from a horse with pathological evidence of viral pneumonia. The Kentucky and Georgia viruses were recovered from nasal swabs collected from vaccinated horses with 4271 -153- V. S. HINSHAW ET AL. fever and nasal discharge. All viruses were isolated in the allantoic cavity of 10-11-day old embryonated chicken eggs (4). When possible, first or second egg passages of field isolates were used for these studies. The viruses were grown in 11-day-old embryonated chicken eggs at 35 °C for 72 h, concentrated by adsorption- elution on chicken erythrocytes, and purified on sucrose gradients (9). Antisera and serological assays Antisera specific for the isolated haemagglutinin (HA) and neuraminidase (NA) of Eq/Miami/1/63 were prepared in goats (17). Post-infection ferret sera were prepared, as already described (10). It should be noted that all the ferrets received an intraperitoneal (i.p.) injection of 5 x 107 EID5o (egg infectious dose) of the same virus at 12 days post- infection and were bled 10 days later. The i.p. boost was necessary to produce a significant antibody response (HI titre > 1:80) in ferrets that had been inoculated intranasally with the equine 2 strains. Haemagglutinin (HA) titrations and haemagglutinin inhibition (HI) tests were performed in microtitre plates with receptor-destroying enzyme-treated sera (10). Neuraminidase titrations (NA) and neuramini- dase inhibition (NI) tests have been fully described (21). Preparation of hybridoma cell lines Hybridoma cell lines were prepared by fusion of the 8-azaguanine-resistant clone of MOPC-21 myeloma cells (P3/X63/Ag8) with spleen cells of immunized Balb/c mice using polyethylene glycol (6), as previously described for influenza virus (8). The mice received two doses of 1000 HA of purified virus, given 2 months apart. The culture fluids from the fused cells were screened by HI tests for the detection of antibodies. Cultures producing antibody to the HA were cloned in soft agar and injected intraperitoneally into pristane-treated mice (8). Ascitic fluid was collected 7-10 days later and used in the assay. RESULTS Antigenic comparison of recent equine 2 isolates from horses in the USA Recent outbreaks of equine 2 viruses in horses in the USA involved both vaccinated and unvaccinated animals. To examine the possibility that antigenic variants may be responsible for these outbreaks, isolates from horses in Kentucky, California and Georgia were compared with the prototype, A/eq/Miami/1/63 (Eq/Miami). In HI tests with ferret antisera (Table 1) the isolates were readily distinguishable from the prototype strain Eq/Miami, as indicated by the > 4-fold difference in HI titres. Ferret antisera to viruses isolated between 1976 and 1981 exhibited similar levels of cross-reactivity among these strains, suggesting that these viruses were closely related; however, these sera reacted poorly, if at all, with Eq/Miami. These results show that antigenic variation was evident in the isolate from 1976, indicating that these changes are not recent events. Similar HI tests with chicken sera and hyperimmune goat and rabbit sera to equine 2 strains (results not shown) did not distinguish between these viruses. This is similar to the findings with influenza viruses isolated from humans, where ferret antisera are needed to distinguish closely related strains within a subtype. Antigenic comparisons between equine 2 viruses isolatedfrom horses in different areas of the world (1963-81) Since similar outbreaks of equine influenza have occurred in Europe, it was of interest to compare the Table 1. Antigenic comparisons of A/Eq/Miami/1/63 with recent H3N8 isolates from horses in the USA HI titresa with ferret antisera to: Virus Eq/Miami/i /63 Eq/Ky/ 1/76 Eq/Ky/ 1/80 Eq/Cal/ 1/80 Eq/Ky/ 1/81 Eq/Miami/1/63 640 20 < 10 < 10 20 Eq/Ky/1/76 20 160 40 40 40 Eq/Ky/ 1/80 40 320 80 40 160 Eq/Cal/ 1/80 80 160 40 80 320 Eq/Ky/ 1/81 40 320 40 40 160 a HI titre = reciprocal of the highest dilution of antisera inhibiting four haemagglutinating doses of virus. 154 ANTIGENIC VARIATION IN EQUINE 2 INFLUENZA VIRUSES American and European isolates to determine if similar viruses were involved. In addition, a number of equine 2 isolates from outbreaks over the last 18 years were included in these studies to examine the possibility that antigenic variants had appeared previously. In HI tests with ferret antisera (Table 2), it is apparent that there are antigenically distinguishable strains of equine 2 influenza A viruses currently circulating in horses in the world. The Eq/Switz/ 2225/79 strain is different from other equine strains isolated in Europe and the USA in 1979-81, e.g., Eq/Switz/1118/79 and Eq/Ky/81. The Eq/Switz/ 2225/79 strain is antigenically similar to the proto- type Eq/Miami while the other current strains, e.g., Eq/Ky/ 1/81 and Eq/Ga/l/81, are more similar to Eq/Uruguay/63 than to Eq/Miami. The equine 2 strains currently circulating in the USA are similar to Eq/Fontainebleu/1/79 but viruses similar to Eq/ Switz/2225/79 have not yet been detected in horses in the USA. It should be noted that the HI tests with ether- treated viruses reduced the ability of ferret antisera to distinguish these strains, which suggests that ether treatment exposes more shared determinants. The reactivity of ether-treated Eq/Miami and Eq/ Table 2. Antigenic comparisons between H3N8 viruse 1963-81 Switz/2225/79 (results not shown) still indicated that these viruses were closely related to each other and distinguishable from other viruses. These results indicate that the majority of equine 2 isolates from recent disease outbreaks possess a haemagglutinin that is antigenically distinguishable from that of Eq/Miami. They also suggest that anti- genic variants co-circulate in the equine population. The other surface antigen, i.e., the neuraminidase (NA), was also examined for evidence of variation. NI tests (Table 3) with ferret antisera to Eq/Miami, Eq/Calif/80, and Eq/Fontainebleu/79 suggested that the NAs of recent isolates were distinguishable from the prototype strain (4-8 fold lower titre). However, in reciprocal tests, ferret antisera to Eq/Miami failed to discriminate between the neuraminadases. Monospecific hyperimmune goat antisera to the NA of Eq/Miami also failed to distinguish the NAs of these 3 viruses. The results suggest that the NAs of recent strains do not differ extensively from the NA of Eq/Miami. Characterization of equine 2 viruses with mono- clonal antibodies to the haemagglutinin. Monoclonal antibodies are useful for detecting variations between closely related influenza viruses (3, 18, 19). 5s isolated from horses in different areas of the world, HI titresa with ferret antisera to: Virus Eq/Miami/i /63 Eq/Uruguay/ 1/63 Eq/Ky/i /76 Eq/Font/i /79 Eq/Switz/2225/79 Eq/Ky/ 1/81 Eq/Miami/l/63 160 80 20 20 40 20 Eq/Uruguay/l/63 40 160 160 80 20 160 Eq/France/67 20 80 160 160 <20 80 Eq/Sao Paulo/69 80 80 40 80 80 40 Eq/Sachyama/7 1 160 80 20 1 60 20 80 Eq/Algiers/72 160 40 20 80 20 40 Eq/Newmarket/76 40 20 20 80 20 40 Eq/Ky/l/76 20 80 160 80 < 20 40 Eq/Kascadeur/78 40 160 320 160 20 80 Eq/Font/79 40 160 320 160 20 80 Eq/Switz/2225/79 40 < 20 < 20 < 20 80 < 20 Eq/Switz/1 1 18/79 40 80 320 80 < 20 80 Eq/Romania/l/80 20 80 160 80 < 20 80 Eq/Cal/1/80 < 20 40 160 40 < 20 160 Eq/Ky/80 40 160 320 320 20 320 Eq/Ky/1/81 20 80 320 80 < 20 160 Eq/Ga/1/81 40 320 640 80 10 80 a HI titre = reciprocal of serum dilution inhibiting four haemagglutinating doses of virus. 155 V. S. HINSHAW ET AL. Table 3. Reactions in neuraminidase-inhibition tests between H3N8 viruses with ferret antisera NI titresa with ferret antisera to: Virus Eq/Miami/ Eq/Fontainebleu/ Eq/Kentucky/ 1/63 79 80 Eq/Miami/1/63 50 20 < 10 Eq/Fontaine- 100 100 35 bleu/79 Eq/Kentucky! 100 75 5080 a NI titre = reciprocal of serum dilution inhibiting 50% of neur- aminidase activity with a reading of approximately 0.600 at 529 nm. Monoclonal antibodies to the haemagglutinins of Eq/Miami/1/63 and Eq/Fontainebleu/79 were used in HI tests (Table 4) against the same panel of viruses examined with ferret antisera (Table 2). Five monoclones to Eq/Miami and two to Eq/ Fontainebleu/79 demonstrated different reactivity patterns with the equine 2 viruses, indicating that these antibodies were recognizing different epitopes and that the equine 2 viruses differed in these epitopes. Reactions with monoclonal antibodies to Eq/ Miami showed that the majority of the recent equine viruses possess the epitopes recognized by these antibodies. The exceptions were Eq/Uruguay/63 and Eq/Algiers/72 which did not react with any of the monoclonal antibodies to Eq/Miami haemag- glutinin. The two earliest isolates, i.e., Eq/Miami and Eq/Uruguay/ 1 /63, were readily distinguishable, indicating that changes in these epitopes are not recent events. With the monoclones to Eq/Fontainebleu/79, the US isolates from 1980-81 reacted to titres similar to the homologous virus, whereas Eq/Miami failed to react with these antibodies. Earlier isolates, such as Eq/Sachyama/71, did react with these monoclones, demonstrating the presence of these epitopes on equine strains before 1979. Using either post-infection ferret sera or mono- clonal antibodies, the recent American viruses and Eq/Fontainebleu/79 were shown to be related to, yet distinguishable from, Eq/Miami. However, isolates such as Eq/Uruguay/63 differ markedly from any of the other viruses based on the results with mono- Table 4. Reactions in haemagglutination-inhibition tests between H3N8 viruses with monoclonal antibodies to the haemagglutinins of A/Eq/Miami/1/63 and A/Eq/Fontainebleu/79 HI titresa with monoclonal antibodies to: Eq/Miami/i /63 Eq/Fontainebleu/79 Virus 1 2 3 4 5 1 3 Eq/Miami/1/63 6 400 6 400 6 400 3 200 6 400 < 100 < 100 Eq/Uruguay/1/63 < 100 < 100 < 100 < 100 < 100 < 100 < 100 Eq/France/67 6 400 400 3 200 3 200 100 < 100 < 100 Eq/Sao Paulo/69 6 400 6 400 6 400 6 400 6 400 < 100 < 100 Eq/Sachyama/71 < 100 < 100 < 100 6 400 < 100 6 400 3 200 Eq/Algiers/72 < 100 < 100 < 100 < 100 < 100 6 400 < 100 Eq/Newmarket/76 6 400 3 200 6 400 3 200 1 600 6 400 6 400 Eq/Ky/76 3 200 100 200 200 100 < 100 < 100 Eq/Kascadeur/78 6 400 400 3 200 400 < 100 6 400 3 200 Eq/Fontainebleu/79 6 400 3 200 3 200 3 200 < 100 6 400 6 400 Eq/Switz/2225/79 6 400 1 600 1 600 6 400 800 < 100 < 100 Eq/Switz/1118/79 3 200 200 3 200 1 600 < 100 < 100 < 100 Eq/Romania/1/80 6 400 400 800 400 < 100 < 100 < 100 Eq/Ky/80 3 200 800 800 1 600 < 100 6 400 800 Eq/Cal/1/80 6 400 3 200 3 200 800 < 100 6 400 1 600 Eq/Ky/1/81 6 400 3 200 3 200 1 600 < 100 6 400 3 200 a HI titre = reciprocal of serum dilution inhibiting four haemagglutinating doses of virus. 156 ANTIGENIC VARIATION IN EQUINE 2 INFLUENZA VIRUSES clonal antibodies, but not with ferret antisera. These discrepancies are most likely a reflection of the high specificity of monoclonal antibodies and of the limited number of monoclonal antibody prepara- tions used in this study. The most striking finding with monoclonal anti- bodies was that the examined equine viruses retained epitopes for 18 years, whereas human strains in the H3 subtype show more variation (19). DISCUSSION The studies reported here were conducted to determine whether antigenic variation could explain the occurrences of recent influenza outbreaks in both vaccinated and unvaccinated horses. Antigenic analyses of equine 2 viruses with ferret antisera and monoclonal antibodies established that, although closely related, the equine 2 viruses are antigenically heterogeneous. The majority of recent isolates are antigenically distinguishable from the prototype, A/Eq/Miami/l/63, but apparently variants co- circulate as demonstrated by the different reactivity patterns of two isolates from horses in Switzerland in 1979. In view of the limited antigenic variation of equine 2 viruses detected in these studies, it is un- likely that the current problems with influenza out- breaks in horses can be solely attributed to antigenic changes in the viruses. The detection of antigenic variation in equine 2 viruses agrees with earlier reports (5, 11, 16). Although equine 2 influenza viruses belong to the H3 subtype, the limited extent of antigenic change in these viruses over an 18-year period contrasts with the more marked variation in human strains (15, 19). In addition, studies on the RNAs of these viruses showed similarities between the isolates. The migration patterns of viral RNAs, separated by polyacrylamide gel electrophoresis (14), showed no differences between Eq/Miami and recent isolates, in contrast to the variety of migration patterns seen with avian (14) and swine (4) viruses. Comparisons of the viruses by hybridization of electrophoretically separated vRNAs with labelled cDNA to Eq/Miami (Naeve, unpublished observations) did not distin- guish between the equine viruses, indicating that all genes of these viruses share > 7OWo homology. These findings suggests that, although antigenic changes are evident, equine strains show surprising conser- vation in view of their continual circulation in nature. It is apparent from the recent problems with influenza in horses that vaccination is not controlling the disease. This poses questions on the efficacy of equine influenza vaccines which have been available since 1964 (2). Two possible explanations are: (a) that current strains differ from Eq/Miami, so the vaccine strain is inappropriate antigenically, and (b) the vaccine potency is inadequate and fails to induce sufficient antibodies to protect against infection with the homologous virus and/or antigenic variants. Although the vaccine has been credited with reducing the infection and severity of disease (12, 13), it has also been noted that the protection is short-lived, requiring revaccination every three months, and that the levels of humoral antibodies are low following primary, as well as subsequent immunization (2). In outbreaks in the USA, some of the horses had been vaccinated regularly every 3 months, yet the HI titres of these horse sera to Eq/Miami were 1:10 to 1:20. In other studies (2), HI titres of > 1:16 prevented infection and disease with Eq/Miami. It is clear that this antibody titre does not prevent infection with the current strains. It seems reasonable, therefore, that the vaccine has to be improved both as regards the strain of virus used and its potency, if vaccination of horses is to be successful. ACKNOWLEDGEMENTS We wish to acknowledge the excellent technical assistance of Meera Krishnan, Phillip Rowley and Jim Wilson. This work was supported by Contract AI-02649 from the National Institute of Allergy and Infectious Diseases, by Cancer Center Support (CORE) grant CA-21765 from the National Cancer Institute, and by ALSAC (St Jude Children's Research Hospital). RESUME ANALYSE DES VARIATIONS ANTIGENIQUES DES VIRUS A EQUINS 2 Des poussees de grippe chez les chevaux, provoquees par des virus equins 2 (sous-type H3N8), deviennent plus fr&- quentes et font souvent des victimes chez des chevaux vaccines. Aussi a-t-on examine une serie de virus grippaux du sous-type H3N8 a la recherche d'eventuelles variations antigeniques dans les isolats de la periode 1963-81. Les 157 158 V. S. HINSHAW ET AL. analyses antigeniques effectuees A l'aide d'anticorps mono- clonaux et de serum de furet preleve A la suite d'une infec- tion ont montre que les hemagglutinines des isolats recents presentaient des differences antigeniques par rapport au prototype A/Miami/1/63 et que plusieurs virus equins 2 de constitution antigenique distincte etaient en circulation dans la population equine. Les analyses ont montre que ces virus, en depit d'une etroite analogie, etaient heterogenes du point de vue antigenique: la majorite des isolats recents se distinguent nettement du prototype A/Miami/l/63; cepen- dant, il existe des variants en circulation simultanee, comme le montrent les schemas de reactivite differents des isolats effectues en Suisse sur des chevaux en 1979. Sur la base de ces etudes, il est recommande d'utiliser une souche equine recente, par exemple la souche A/Fontainebleau/ 1/79 ou la souche A/Kentucky/1/81, comme prototype complemen- taire a l'interieur de ce sous-type. 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