Experimental infections in man and horses with influenza A viruses*

Bull. Org. mond. Sante 1969, 41, 447-452 Bull. Wid Hith Org. Experimental Infection in Man and Horses with Influenza A Viruses* J. A. KASEL I & R. B. COUCH2 The recognition of an antigenic relationship between the haemagglutinins of A/Equi-2 and A2/Hong Kong/68 viruses led to experimental studies in man and horses with these virus types. Human volunteers were inoculated with A/Equi-2/Miami/63 virus and virus shedding ensued in all subjects. The most common clinical response was a febrile illness indistin- guishable from naturally occurring human influenza. After administration of A2/Hong Kong/68 virus to 10 ponies there was virus shedding from 9 and a febrile response in 6. When the human subjects previously inoculated with equine virus were challenged uith A2/Hong Kong/68 virus, the frequency of illness and the extent of virus shedding were lower than was observed among control individuals. This immunity wasfound to be related to the level of heterologous serum antibody to the human virus which developed after equine virus infection. Challenge with A/Equi-2/Miami/63 virus of ponies previously inoculated with A2/Hong Kong/68 virus, in the absence of any measurable levels of heterologous antibody to the human strain, resulted in less shedding of virus among these than occurred in control animals. An antigenic relationship has been established between the haemagglutinins of A/Equi-2 virus isolated from horses in 1963 and a type A2 virus which caused epidemic illness in human populations during 1968 and 1969 (Coleman et al., 1968; Kasel, Fulk & Couch, 1969). The detection of this relation- ship suggested that strains from one of the species may have been originally derived from the other species. In order to evaluate this possibility and to determine whether the serological relationship had biological significance, experimental studies in man and horses with A/Equi-2 virus and the new human A2 variant were performed. In man, challenge studies were conducted to assess the protective effect of heterologous serum antibody induced by prior infection with equine influenza virus (Couch et al., * These studies were supported in part by research con- tract PH 43-68-936, Vaccine Development Branch, National Institute of Allergy and Infectious Diseases, National Insti- tutes of Health, Bethesda, Maryland 20014, USA. I Head, Medical Virology Section, Laboratory of Clinical Investigation, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland 20014, USA. ' Associate Professor, Departments of Microbiology and Medicine, Baylor College of Medicine, Houston, Texas 77025, USA. 1969).3 In horses, studies were done to assess the effect of inoculation with the human influenza virus on subsequent challenge with equine virus (Kasel, Fulk & Harvey, 1969). The purpose of this report is to summarize the results of these studies. MATERIALS AND METHODS Subjects Subjects were adult male volunteers between the ages of 21 and 35 years who lacked detectable serum neutralizing antibody (<1 : 2) to A Equi-2, Miami/63 and had low or no detectable titre to A2/Hong Kong/68 viruses. Medical examinations to ensure good health were performed and written informed consent was obtained prior to viral inoculations. Horses used in these studies were Chincoteague ponies (Equus cabullus) selected from a wild herd on Assateague Island, Virginia. The animals were obtained from a population which was relatively isolated from human beings and had no known previous contact with other horses. The ponies ' Couch, R. B., Fulk, R. V., Douglas, R. G., Jr, Kasel, J. A. & Knight, V. Unpublished data. 2388 -447- J. A. KASEL & R. B. COUCH lacked measurable serum neutralizing antibody (<1: 2) to the challenge viruses and were approxi- mately 8 months of age at the time of inoculation. Inoculum and method of inoculation The Miami/63 strain of A/Equi-2 virus isolated from a naturally infected horse and a strain of A2/Hong Kong/68 isolated from 2 different persons were used to prepare inoculum pools by previously described procedures (Couch et al., 1969; Cameron et al., 1967; Kasel, Fulk & Harvey, 1969). Embryo- nated hens' eggs, hamster kidney (HK), African green monkey kidney (AGMK), and human em- bryonic kidney (HEK) were used to prepare viral inocula. The passage history of the A/Equi-2 virus inoculum pool used in human volunteer experiments was egg5, HK2, man,, HK4, man1, HEK3, and of that used in pony experiments was egg5, HK2, pony1, AGMK1, HEK2, pony1, AGMK2, pony1, HEK2, pony1, HEK3, pony1, HEK3, pony1, HEK2. Pool 1 and pool 2 of A2/Hong Kong/68 virus were tissue- culture fluid harvests from the sixth and second viral passages in HEK cultures. The human volunteers were given A/Equi-2/Miami/63 or A2/Hong Kong/68 viruses by spray into the nasopharynx and/or by nasal instillation. The animals were given viral inoculum according to the following methods. A2/Hong Kong/68 (pool 1) was sprayed into the nasopharynx by nebulization and also injected into the tracheal lumen by syringe and needle; A2/Hong Kong/68 (pool 2) was sprayed into the nasopharynx and oropharynx by nebulization; and A/Equi-2/ Miami/63 was sprayed into the nasal cavities by nebulization. Clinical and laboratory methods The procedures employed for clinical evaluation of subjects, virus isolation, and serum neutralizing antibody titres have been described elsewhere (Kasel, Fulk & Harvey, 1969; Couch et al., 1969). RESULTS Response ofman to equine influenza virus The illness responses of human volunteers follow- ing nasopharyngeal inoculation with 108.75 50% tissue-culture infectious doses (TCID50) of A/Equi-2 virus are shown in Table 1. Thirteen of 15 individuals developed illness on the second or third day after inoculation. The most common clinical response was a febrile upper respiratory and systemic illness which lasted 2-4 days; 8 subjects showed this type of TABLE I ILLNESS RESPONSE OF ADULT MALE VOLUNTEERS FOLLOWING NASAL INOCULATION WITH A/Equi-2/Miami/63 INFLUENZA VIRUS of men Type of illness 8 Febrile upper respiratory and systemic 2 Febrile upper and lower respiratory and systemic 3 Afebrile upper respiratory 2 None illness response. Two volunteers also developed significant symptoms of lower respiratory illness which included paroxysmal episodes of cough, sub- sternal discomfort and tenderness of the trachea. One of these individuals also developed an identical syndrome 10 days after inoculation. Three persons had an afebrile upper respiratory illness only. When symptoms and signs were tabulated and frequencies compared with those described for naturally occur- ring type A influenza illness (Stuart-Harris, 1965), it was observed that the febrile response was of shorter duration (mean, 1.3 days), nasal discharge and obstruction were slightly more frequent, and there was a slightly less frequent occurrence of cough and sore throat among the subjects inoculated with equine influenza virus. These differences may be attributable to the route of administration of equine virus since it is known that the majority of the virus dose was deposited in the nasopharynx. Never- theless, the syndrome of sudden onset of febrile systemic illness with rhinitis and cough, the most common clinical response observed in the volun- teers, is compatible with clinical descriptions of naturally occurring human influenza. Virus shedding occurred in each of the 15 volun- teers. Isolations were obtained beginning on day 1 from 5 men and from all others between days 2 and 4 after inoculation. The duration of virus shedding was limited to the first 6 days except for 4 individuals who yielded a virus-positive specimen on day 10. Fourteen of the 15 subjects exhibited a 4-fold or greater serum neutralizing antibody response to the infecting virus and, of these, 9 developed heterolo- gous serum neutralizing antibody to A2/Hong Kong/68 influenza virus. All individuals were initially free of measurable antibody to each virus 448 EXPERIMENTAL INFECTION WITH INFLUENZA A VIRUSES type except for 4 volunteers who had low titres (1: 2 or 1: 4) to the human strain. The relationship between antibody responses to A/Equi-2 and A2/Hong Kong/68 viruses is shown in the accom- panying figure. The 28-day post-inoculation serum antibody rises to the equine and human strains ranged from l : 2 to >1: 512 and <1 : 2 to l: 4096, respectively. A comparison of the magnitude of in- creases in antibody levels to each of the viruses revealed that persons with a high increase in antibody to A/Equi-2 virus tended also to exhibit a high rise in antibody to the human influenza virus (r= +0.477, P<0.05, Spearman rank correlation test). Response ofponies to human influenza viruses Responses of the ponies to inoculation with A2/Hong Kong/68 influenza virus are presented in Table 2. For comparative purposes, responses of 5 animals to inoculation with an antigenically dis- similar human-type A2 virus are also included (Kasel, Fulk & Harvey, 1969). Among the 10 ponies inoculated with A2/Hong Kong/68 virus, a febrile response occurred in 6, virus shedding from 9 and a serum neutralizing antibody response in 4. In con- trast, among the 5 animals administered the A2/ Rockville/65 influenza virus, a human strain known not to possess a virion surface antigen related to A/Equi-2 virus, there were no measurable responses. Fever was first observed in ponies about 30 hours after virus inoculation and thereafter ranged from 102.5°F to 104.2°F (39.20C-40.10C) for 1-3 days. Six-hour post-inoculation specimens obtained from all ponies were negative for virus. Isolations were first obtained from 5 ponies on day 1, from 3 on day 2 and from 1 on day 3. Three of the animals shed virus for 1 day, 3 for 2 days, 2 for 3 days, and 1 for 5 days. SERUM NEUTRALIZING ANTIBODY TITRES TO A/Equi-2/Miami/63 AND A2/Hong Kong/68 VIRUSES IN HUMAN ADULT VOLUNTEERS 28 DAYS FOLLOWING CHALLENGE WITH A/Equi-2/Miami/63 VIRUS 14r U, D 12 60 co"I I z 0 U,O oU LL 0 _ 6- ° 4 z D 2 cc U) ) .0 S 0 I 0 L I . 0 0 0 2 4 6 8 10 I SERUM ANTIBODY RISE TO A/EQUI-2/MIAM,/63 VIRUS (FOLD INCREASE) The serum antibody titres to A2/Hong Kong/68 virus ranged from 1: 2 to 1: 8 in the 4 ponies with a serological response. No heterologous antibody responses to A/Equi-2 virus were detected. Challenge ofhuman volunteers with A2/Hong Kong/68 virus Four weeks after inoculation with A/Equi-2 virus all 15 of the infected volunteers and 19 serum-anti- TABLE 2 RESPONSES OF CHINCOTEAGUE PONIES FOLLOWING INOCULATION OF A2/Hong Kong/68 INFLUENZA VIRUS Administered Nof No. with No. wit No. with Virus inoculum dose No f febrile virusth 2-fold(TCIDso) animals illness lations antibody (>102.50F) a ISOlaios rise A2/Hong Kong/68 (pool 1) 10' 5 5 4 5 3 A2/Hong Kong/68 (pool 2) 106.2 5 2 4 1 A2/Rockville/65 I0' ' 5 0 0 0 a Normal equine temperature is up to 102.5°F (39.2°C). 2 X XAA9,_ J. A. KASEL & R. B. COUCH TABLE 3 OCCURRENCE OF ILLNESS, DISTRIBUTION OF VIRAL ISOLATES AND SERUM ANTIBODY RESPONSES OF ADULT MALE VOLUNTEERS FOLLOWING NASAL INOCULATION OF A2/Hong Kong/68 INFLUENZA VIRUS No. of men with indicated response to virus challenge Challenge group |Ngroup No. with No. with indicated number of viral isolates No. withgroup No. with ~~~~~~~~~~~~~>4-foldillness 0 1 2 3 4 5 6 7 antibody rise Prior inoculation with A/Equi-2/Miami/63 15 1 10 2 2 0 0 0 1 0 4 Controls 19 9 8 1 0 1 3 2 3 1 14 body-negative volunteers were simultaneously chal- lenged intranasally with 103.2 TCID50 of A2/Hong Kong/68 virus (pool 2). The responses to these inoculations are summarized in Table 3. Administra- tion of the human strain to subjects previously in- oculated with A/Equi-2 virus was followed by illness in 1, virus shedding in 5 and serum antibody rise in 4. The relative frequency of illness responses among subjects previously given equine virus tended to be less than that seen among the control in- dividuals (P<0.06). The difference in the distribu- tion of viral isolates in the equine viral group and that in the control group challenged with the same human strain was clearly significant (P<0.05). As shown in Table 4, the resistance to challenge was found to be related to the titre of serum antibody to A2/Hong Kong/68 virus which developed after in- fection with equine influenza virus. At the time of inoculation with the human strain, 6 volunteers lacked measurable antibody to this strain, 5 exhibited low levels and 4 had high titres. Infection as mea- sured by illness, virus shedding and/or rise in serum antibody titre occurred in 6 subjects. Four of the 6 with no detectable antibody to the challenge virus TABLE 4 OCCURRENCE OF INFECTION AMONG ADULT MALE VOLUNTEERS FOLLOWING NASAL INSTILLATION OF A2/Hong Kong/68 INFLUENZA VIRUS ACCORDING TO PRE-INOCULATION ANTIBODY TITRE Pre-inoculation serue No. of volunteers No. infected <2 6 4 4-32 5 2 256-4 096 4 0 a Reciprocal neutralization titre to A2/Hong Kong/68 virus. became infected with this strain, 2 of 5 with inter- mediate titres became infected, and none of 4 with high titres. The decrease in the occurrence of infec- tion in relation to the titre of serum antibody was statistically significant (P<0.05, Yates mean score test). Challenge ofponies with A/Equi-2/Miami/63 virus Seventy-five and 52 days after inoculation of ponies with A2/Hong Kong/68 virus, pools 1 and 2 respectively, these 10 animals and 5 serum-antibody negative controls were given 104-0 TCID50 of A/Equi-2/Miami/63 virus. The occurrence of febrile responses and patterns of virus shedding are shown in Table 5. Two ponies that had been previously exposed to pool 2 of A2/Hong Kong/68 virus and one control animal developed a febrile response. Virus shedding of A/Equi-2 virus was less in the previously challenged animals than in the 5 control animals. The difference in the distribution of viral isolates from ponies that had been given pool 1 and from control animals was statistically significant (P<0.02). However, the difference was not significant when ponies inoculated with pool 2 and controls were compared (P>0.10). The geometric mean serum neutralizing antibody titres to A/Equi-2 and A2/Hong Kong viruses before and after challenge with equine influenza virus are shown in Table 6. At the time of virus challenge there was no measurable antibody to the equine strain and only low levels to the human strain in the animals that received a prior challenge with A2/ Hong Kong/68 virus. Following challenge, each of the 10 animals previously inoculated with A2/Hong Kong/68 virus exhibited a serum antibody rise to both the equine and human strains. All control ponies developed an antibody response to A/Equi-2 virus and of these, 3 showed a rise in titre to A2/Hong Kong/68 virus. The post-challenge geometric mean 450 EXPERIMENTAL INFECTION WITH INFLUENZA A VIRUSES TABLE 5 OCCURRENCE OF FEVER AND DISTRIBUTION OF VIRAL ISOLATES OF CHINCOTEAGUE PONIES FOLLOWING NASAL INOCULATION OF A/Equi-2/MiamiJ63 INFLUENZA VIRUS No. cof No. of animals No. of animals with the indicated Challenge group animals with febrile number of isolatesChallengg inimalsp responsengroup (>,102.52F) 0 1 2 3 4 5 Prior inoculation with A2/Hong Kong/68(pool 1) 5 0 2 2 1 0 0 0 Prior inoculation with A2/Hong Kong/68 (pool2) 5 2 3 0 0 1 1 0 Controls 5 1 0 1 0 2 1 1 antibody titres to A2/Hong Kong/68 virus among both groups of ponies previously inoculated with that type were significantly higher than those seen in the control animals (P<0.01 for pool 1 and P<0.02 for pool 2). The same type of comparison for A/Equi-2 responses showed the mean antibody titre in animals inoculated with only pool 1 to be signifi- cantly greater (P<0.01). In addition, the mean antibody titre to the equine and human influenza viruses tended to be higher among the ponies that received inoculum pool 1 than among those that received pool 2, and in the case of A/Equi-2 virus, the difference in mean antibody titre was highly significant (P<0.01). TABLE 6 SERUM NEUTRALIZING ANTIBODY RESPONSES OF CHINCOTEAGUE PONIES FOLLOWING NASAL INOCULATION OF A/Equi-2/Miami/63 INFLUENZA VIRUS Geometric mean antibody titre a, b No. of Challenge group animals A/Equi-2/ A2/Hong in group Miami 63 Kong/68 Si S2 Si S2 Prior inoculation with 5 <2 84.5 1.7 73.5 A2/Hong Kong/68(pool 1) Prior inoculation with A2/Hong Kong/68(pool 2) 5 <2 8.0 1.5 24.3 Controls 5 <2 4.6 <2 1.7 a S refers to serum specimen collected at the time of chal- lenge with A/Equi-2 virus and S2 to specimen obtained 25 days after challenge. b Titre expressed as reciprocal of serum dilution. DISCUSSION The results reported in the present study indicate that A/Equi-2 influenza virus is capable of infecting man and producing an illness which is indistinguish- able from naturally occurring human influenza. The rates of infection and illness in human volunteers. given A/Equi-2 virus in the study reported here were higher than those observed in other experiments in man using strains of the same virus type (Alford et al., 1967). Among 33 subjects to whom A/Equi-2 viral inocula had been administered, illness occurred in 4 persons, virus was recovered from 21 and rises in serum antibody were observed in 20. Included in this study group were 6 subjects who had been given the same inoculum as that used in the study described in this report. Following inoculation, there was no illness in any of the individuals, although virus was isolated from 4 and antibody responses occurred in 5. The reasons for these differences in illness response are not known at this time. The reported results also indicate that A2/Hong Kong/68 influenza virus is capable of infecting horses and producing a febrile illness. Of particular interest are the contrasting results in ponies inocu- lated with A2/Rockville/65 or A2/Hong Kong/68 viruses. A possible explanation for occurrence of viral infection in the latter ponies and not in those- given A2/Rockville/65 virus is the difference in sensitivity to an inhibitor in normal equine serum. It. was observed that the A2 strain isolated in 1965 was. neutralized in vitro by the inhibitor, whereas the 196& variant was unaffected (Kasel, Fulk & Harvey, 1969). The known antigenic relationship between A/Equi-2 and A2/Hong Kong/68 viruses was shown to have biological relevance in that human subjects 451 452 J. A. KASEL & R. B. COUCH who were given equine virus and developed serum antibody to A2/Hong Kong/68 virus were protected against a later challenge with the human strain in relation to the magnitude of heterologous antibody that developed. Earlier studies have shown that heterologous serum antibody responses to strains of human type A2 virus occur in horses naturally in- fected with A/Equi-2 virus (McQueen et al., 1969). This would provide for an additional barrier against transmission of human strains to horses. Moreover, the development of heterologous serum antibody to human type A2 viruses in man or horses exposed to A/Equi-2 virus suggests that surveillance data based on serological surveys alone may not be a reliable indicator of interspecies spread during outbreaks of influenza in either species. This would also be true for epidemics caused by A2/Hong Kong/68 virus. Immunity against A/Equi-2 virus challenge of 2 groups of ponies previously inoculated with the human strain was limited to a reduction of virus shedding in one group of animals. Thus, little to no protection of ponies was afforded by the prior infec- tion with A2/Hong Kong/68 virus. Since no de- tectable heterologous serum antibody developed in these animals, we suggest that this is the reason infection ensued following A/Equi-2 virus challenge. In this respect, the ponies were similar to humans who did not develop heterologous antibody in that the occurrence of infection and illness in volunteers without antibody was similar to that which developed in controls. The presence of A/Equi-2 virus in equine popula- tions for at least 5 years prior to the occurrence of epidemic influenza by A2/Hong Kong/68 virus and the available epidemiological data (Davenport, Hennessey & Minuse, 1967) suggest that horses are not active reservoirs of virus for man. Nevertheless, the appearance in man and horse of type A influenza viruses that are antigenically related suggests that an exchange of viruses between these hosts may on occasion occur in nature. The data presented in this report tend to support that suggestion. REFERENCES Alford, R. H., Kasel, J. A., Lehrich, J. R. & Knight, V. (1967) J. Epidem., 86, 185-192 Cameron, T. P., Alford, R. H., Kasel, J. A., Harvey, E. W., Byrne, R. J. & Knight, V. (1967) Proc. Soc. exp. Biol. (N.Y.), 124, 510-515 Coleman, M. T., Dowdle, W. R., Pereira, H. G. & Schild, G. C. (1968) Lancet, 2, 1384-1413 Couch, R. B., Douglas, R. G., Jr, Kasel, J. A., Riggs, S., & Knight, V. (1969) Nature (Lond.) (in press) Davenport, F. M., Hennessey, A. V. & Minuse, E. (1967) J. exp. Med., 126, 1049-1061 Kasel, J. A., Alford, R. H., Knight, V., Waddell, G. H. & Sigel, M. M. (1965) Nature (Lond.), 206, 1-6 Kasel, J. A., Fulk, R. V. & Couch, R. B. (1969) J. Immu- nol., 102, 530-532 Kasel, J. A., Fulk, R. V. & Harvey, E W. 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