Infection with A2 Hong Kong influenza virus in domestic cats*

Bull. Org. mond. Sante 1970, 43, 859-862Bull. Wid Hithi Org.J Infection with A2 Hong Kong Influenza Virus in Domestic Cats* C. K. J. PANIKER 1 & C. M. G. NAIR 2 The antigenic relationship ofA2 Hong Kong influenza virus with equine influenza virus, and its ability to infect horses and baboons, have led to studies on the susceptibility of domestic animals to the virus. In this study it was found that cats could be infected with A2 Hong Kong influenza virus by intranasal inoculation or by contact with an infected cat or with a human influenza patient. There was no clinical illness, but infected animals shed the virus from the throat for I week and developed haemagglutination-inhibiting antibodies. A survey of normal cat sera showed that 6 out of 28 sera inhibited haemagglutination by A2 Hong Kong influenza virus. The results suggest that domestic cats may act as vectors in the transmission ofinfluenza virus. Experimental infection in cats may be used as a laboratory model for influenza. There has been much speculation about the possible role of animals in the epidemiology of human influenza. It has been suggested that pan- demic strains of human influenza virus may perhaps originate in animals (Andrewes, 1959; Fenner, 1968). Many animals and birds have been shown to suffer from natural influenza, and porcine, equine and avian influenza viruses have been isolated and char- acterized (Andrewes, 1964). Antigenic relationships have been established among the haemagglutinins and neuraminidases of some human, avian and animal influenza viruses (Pereira, Tumova & Webster, 1957; Webster & Pereira, 1968; Kasel, Fulk & Couch, 1969). The experimental production of recombinants between human and animal influenza viruses, besides indicating their common ancestry, suggests that similar hybrids may occur in nature also (Tumova & Pereira, 1965; Easterday et al., 1969). While no interspecies transfer of influenza infection has been convincingly demon- strated in nature, it has been shown that man may be experimentally infected with equine influenza virus, and horses and baboons with human influenza A2 virus (Kasel & Couch, 1969; Kalter et al., 1969). The demonstration of infection by human influenza viruses in an animal species in close contact with * From the Medical College, Calicut-8, Kerala, India. Professor of Microbiology. Assistant Professor of Microbiology. man would perhaps be more relevant in under- standing the role of animals in human influenza. We report the susceptibility of domestic cats to A2 Hong Kong influenza virus. MATERIALS AND METHODS Virus strains Influenza virus strains isolated in this laboratory were used. Throat washings from influenza patients were divided into aliquots and stored at-30°C. Virus isolation was by direct allantoic inoculation into chick embryos (Paniker & Nair, 1969). The isolates were shown to be antigenically identical with A2/Hong Kong/68 influenza virus by com- parison with standard strains obtained from the Government of India Influenza Centre, Pasteur Institute, Coonoor, South India. Animals and animal inoculation procedures The animals used were adult domestic cats (Felis catus L.) and kittens under 8 weeks of age housed in large wire cages. In contact experiments, the cages were kept 1 ft (30.5 cm) apart. For experimental infection, approximately 0.2 ml of virus-infected allantoic fluid from the first egg passage (haemagglutinin (HA) titre ranging from 20 to 80) was inoculated intranasally into cats under light ether anaesthesia, within a few hours after collection. Blood samples were collected by 2607 859 C. K. J. PANIKER & C. M. G. NAIR cardiac puncture before and after infection, and the rectal temperature was recorded. For virus isolation by direct allantoic inoculation, throat washings were collected periodically after infection from the cats under anaesthesia. Haemagglutination inhibition (HI) test All sera were treated with 0.011 M potassium periodate and heated at 56°C for 30 min prior to testing. Previous tests had shown this to be the most effective method for removing nonspecific inhibitors to A2 Hong Kong virus in cat sera. For the survey of HI antibodies in normal cat sera, combined treatment with trypsin and periodate (Ananthanarayan & Paniker, 1960) was also used for inactivation of nonspecific inhibitors. HI tests were done in plastic trays using 4 units of virus and 0.5 % fowl erythrocytes. The tests were run in parallel using A2/Hong Kong/68 influenza virus and the locally isolated strains used for infection. In no case was a difference in titre greater than 2-fold observed between the two. Since 3 different strains had been used for infection in different experiments, only HI titres against A2/Hong Kong/68 virus are shown in the results. All titres are expressed as reciprocals of the dilution. RESULTS Experimental infection of kittens Two kittens (no. 1 & 2), both 3 weeks old, were inoculated intranasally with virus-infected allantoic fluid and housed in one cage. Serial throat washings were collected, and blood samples were obtained before and 15 days after infection. Virus could be recovered from both animals, the period of virus shedding lasting for up to 6 days; no virus could be isolated 13 days after infection. The kittens had no antibodies to the virus before infection, but post- infection sera had HI antibody titres of 160 and 320. Transmission of infection to cage mate A kitten (no. 3) from the same litter was left as a contact in the same cage. Serial throat washings and paired serum samples were collected. Virus was recovered from the contact kitten for up to 6 days (on day 1 and day 6, but not on day 4) and the HI antibody titre rose from <10 to 120. Reinfection in kittens Kittens no. 1, 2 and 3 were reinfected with the same virus 2 months after the initial infection. No virus was isolated from serial throat washings, but the HI antibody titre increased from 80, 80 and 20, to 640, 160 and 1280, respectively, 12 days after infection. Experimental infection in adult cats Two adult cats (no. 4 & 5) were intranasally infected with the virus. Both animals shed the virus and they developed HI antibody titres of 640 and 320, respectively. Transmission of infection to animals in adjacent cages For these experiments, infected and contact ani- mals were kept in separate cages 1 ft (30.5 cm) animals were kept in separate cages 1 ft (30.5 cm) apart. One kitten (no. 6), 3 weeks old, was intranas- ally infected and another (no. 7), 6 weeks old, was left in the adjacent cage. The contact kitten shed the virus (on day 5 and day 8 after exposure) and developed an HI antibody titre of 320. Two adult cats (no. 8 & 9) were left as contacts in separate cages adjacent to animals no. 4 and 5, from the time the latter were infected. Virus could be recovered from one of the contact animals (on days 3 and 5 after exposure), which also developed an HI antibody titre of 640. The other animal did not shed the virus, though it developed an HI anti- body titre of 20. Transmission of infection by contact with influenza patient Two kittens (no. 10 & 11), 6 weeks old, under light ether anaesthesia were directly exposed for about 2 min to a patient suffering from influenza, identified by virus isolation and serology, on the first day of his illness. The patient was asked to cough and sneeze so that the animals were exposed to the spray of droplets. One of the kittens (no. 10) did not develop antibodies and no virus could be recovered from its throat, but the other (no. 11) shed the virus up to 8 days (on days 3 and 8, but not on days 1 and 5) after contact and developed an HI antibody titre of 80. HI antibodies in normal cat sera Sera from 28 cats, 15 adults and 13 kittens, were tested for HI antibodies after treatment with per- iodate alone and with periodate and trypsin. The results were not significantly different after the two procedures. Inhibition of haemagglutinin was seen in 7 sera (see accompanying table). 860 A2 HONG KONG INFLUENZA VIRUS IN DOMESTIC CATS 861 RESULTS OF Hi TESTS WITH A2IHONG KONG/68 INFLUENZA VIRUS AND NORMAL CAT SERA No. of sera with Hi titres nim als otal|<10 10 | 20 40 80 160 320 Adult cats 11 0 0 0 1 1 2 15 Kittens 10 1 0 0 0 2 0 13 Total 21 1 0 0 1 3 2 28 DISCUSSION The results reported show that cats are susceptible to A2/Hong Kong influenza virus infection. Not only can they be infected by intranasal challenge, but they also transmit the infection to contacts kept either in the same cage or in neighbouring cages. The infected animals showed no evident clinical illness. There was no rise of temperature, nor was there any observable discharge from the nose, coughing or sneezing. But the virus was shed from the throat for at least 1 week after infection and HI antibodies were produced. Infection appeared to produce local immunity, as after reinfection no virus could be recovered from the throat, though there was an increase of antibody titre. Of particular interest was the finding that one of two cats exposed to a human influenza patient developed the infection, indicating direct interspecies transfer of the virus. This may be of epidemiological significance and suggests that cats, because of their close contact with man, may act as vectors in the chain of virus transmission. If transmission of infection from man to cats were a common event in nature, cat serum surveys should reveal antibodies to human influenza viruses. But most animal serum surveys have not included cats. Meenan, Boyd & Mullaney (1962) found that 4 out of 20 cat sera inhibited haemagglutination by A2/ Asia/57 virus. Though the sera were pretreated with periodate, they ascribed this to non-specific inhibitors as there was no inhibition of haemag- glutination by an inhibitor-insensitive strain. Out of 28 cat sera tested in this study, 6 inhibited hae- magglutination in high titres (>80); 4 out of 15 adults and 2 out of 13 kittens. However it has not been established whether these represent antibodies or non-specific inhibitors. The susceptibility of cats to influenza virus may have other applications. The need for a convenient laboratory model for influenza has long been felt. Mice have been used for studying the experimental epidemiology of influenza (Schulman, 1998), and cats may provide an alternative model. RESUME INFECTION PAR LE VIRUS GRIPPAL A2 HONG KONG CHEZ LE CHAT DOMESTIQUE On a inoculd par voie intranasale a des chats domes- tiques un virus grippal isold chez 1'homme et offrant les memes caracteristiques antig6niques que le virus A2/Hong Kong/68. Les animaux n'ont presente aucun signe clinique de maladie, mais ils ont 61imin6 le virus dans les secretions pharyng6es pendant une semaine environ et ont reagi par la production d'anticorps inhibiteurs de 1'hemagglutina- tion. Des chats inocules ont transmis l'infection a des congeneres places dans la meme cage ou dans une cage voisine. Sur deux chatons mis en contact pendant 2 minutes avec un malade atteint de grippe, un a con- tracte l'infection, avec excretion de virus pendant 8 jours et e1aboration d'anticorps IH. Par ailleurs, sur 28 s6rums preleves chez des chats bien portants, 6 inhibaient 'he'magglutination par le virus A2/Hong Kong/68. On n'a pu cependant etablir si le phenomene etait diu a la presence d'anticorps ou a I'action d'inhibiteurs non specifiques. Ces observations demontrent la receptivite du chat domestique au virus grippal humain. Outre leur interet 6pidemiologique, elles donnent a penser que le chat pourrait servir de modele pour 1'etude de l'infection grippale au laboratoire. REFERENCES Ananthanarayan, R. & Paniker, C. K. J. (1960) Bull. Wid Hlth Org., 22, 409-419 Andrewes, C. H. (1959) Perspect. Virol., 1, 185 Andrewes, C. H. (1964) Viruses of vertebrates, London, Bailliere, Tindall and Cox, pp. 110-114 Easterday, B. C., Laver, W. G., Pereira, H. G. & Schild, G. C. (1969) J. gen. Virol., 5, 83-91 Fenner, F. J. (1968) The biology of animal viruses, New York and London, Academic Press, vol. 2, p. 775 862 C. K. J. PANIKER & C. M. G. NAIR Kalter, S. S., Heberling, R. 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