Intervirology 6 : 315-318 (1975/76)
Demonstration of Antibodies to Both Hemagglutinin and Neuraminidase Antigens of H3N2 Influenza A Virus in Domestic Dogs E dwin D. K ilbourne and J. M ichael K ehoe Mount Sinai School of Medicine, City University of New York, N.Y.
Key Words. Influenza • Animal influenza • Canine influenza • Neuraminidase • Hemag glutinin Summary. Serologic evidence of infection with human (H3N2) influenza viruses of 6 of 79 dogs sampled in New York City in March-April 1973 was obtained through the use of four different methods for measurement of anti-hemagglutination and anti-neuramini dase antibody.
Although many animals are susceptible to experimental infection with influenza viruses, natural infection has been documented in only a limited number of species [1], In view of current speculation that animals may serve as a reservoir for pandemic variants of influenza A viruses [2,3], evidence of natural infection in domestic animals with human influenza viruses has con siderable epidemiologic importance. Experimental infection with both influ enza A and B viruses has been produced in domestic dogs (reviewed by N ikitin et al. [4]). However, little evidence has been provided for natural in fection of this species. N ikitin et al. [4] sampled sera from 271 domestic dogs following H3N2 influenza epidemics in the United States and United King dom, and demonstrated antibody to A/Hong Kong/6/68 virus in 5.9% of animals. Reports from the Soviet Union (cited in Easterday and C ouch [1]) noted that canine illnesses and death in the Vladivostok area increased in assoAddress inquiries to: Dr. E. D. K ilbourne, Department of Microbiology, Mount Sinai School of Medicine, Fifth Avenue and 100th Street, New York, N Y 10029 (USA)
Downloaded by: HSC Library-Serials Dept., SUNY 129.49.5.35 - 11/28/2017 3:35:54 PM
Received: February 16, 1976.
316
K ilbourne/K ehoe
dation with influenza epidemics in man, in February 1970 and December 1971, and that Hong-Kong-Iike influenza viruses were isolated from dogs dur ing those periods. However, definitive serologic evidence for infection with a specific influenza virus rests on demonstration of antibody to both hemag glutinin and neuraminidase antigens, particularly in the case of animal influ enza A viruses in which hemagglutinin and neuraminidase appear in varying combinations [5], Serologic tests used in previous studies [4], ‘hemadsorptionneutralization’ and complement fixation, do not distinguish between anti body to hemagglutinin and to neuraminidase and probably are mediated principally through the hemagglutinin antigen. Therefore, it seems appro priate to add further evidence to that already reported on infection of do mestic dogs with influenza. In the present study, we have employed serologic techniques capable of independent assessment of both anti-hemagglutinin and anti-neuraminidase responses. Single serum specimens from 79 dogs in the New York metropolitan area were obtained randomly from clinic visits during March and April of 1973. Animals were unselected and their history with respect to possible influenza exposure was not known. Anti-hemagglutinin antibody was measured in receptor-destroying-enzyme (RDE)-treated sera by conventional hemag glutination inhibition (HI) tests as previously described [6], employing A/ England/42/72 and A/Aichi/68 (H3N2) influenza viruses. Sera with which inhibitory activity was demonstrated were also titrated in plaque neutraliza tion [plaque inhibition (PI)] tests [7] against recombinant virus A/England/42/ 72-Prague/l/56 (H3i:ngNeql) to measure antibody specific for the hemag glutinin. As noted in table I. positive HI tests against one or the other antigen were observed in 10 of 79 dogs studied, with demonstration of higher titers against the 1968 virus in all but one instance. Nonspecific serum inhibitors have often obfuscated interpretation of HI tests in surveys of animal sera for influenza antibody [8]; however, neutralization is effected only by antibody except in the case of certain inhibitor-sensitive *+ ’ H2N2 strains [9], There fore, it was important to show that of the 10 sera positive by HI, 7 had signifi cant levels of anti-H3 neutralizing (PI) antibody. Further evidence for infection of the animals with H3N2 virus was pro vided by demonstration of anti-neuraminidase (anti-N2) antibody in the sera of 6 dogs by specific inhibition of viral neuraminidase (Nl [6]) (table I). In 2 of these dogs, neuraminidase antibody was demonstrated also by plaque-size reduction (PSR) [7] of the recombinant virus H0pn8N2j3ng that possesses only the N2 antigen of H3N2 virus and hence is a neuraminidase-specific serologic reagent. This virus was also employed in NI tests.
317
Influenza in Dogs
Table /. Demonstration in canine sera of antibodies reactive with hemagglutinin and neu raminidase antigens of influenza A virus of H3N2 subtype Dog
Anti-hemagglutinin antibody hemagglutination inhibition H3N2(Eng/72) H3N2(Aichi/68)
20a 24a 2 II 22 25 26a 3 32 20
20" 10 20 10 20 160 it8N 2Eng
PSRC H0pBsN2Bng
400 400 400 800 6,400
< 400 < 400 < 400
Demonstration of Antibodies to Both Hemagglutinin and Neuraminidase Antigens of H3N2 Influenza A Virus in Domestic Dogs E dwin D. K ilbourne and J. M ichael K ehoe Mount Sinai School of Medicine, City University of New York, N.Y.
Key Words. Influenza • Animal influenza • Canine influenza • Neuraminidase • Hemag glutinin Summary. Serologic evidence of infection with human (H3N2) influenza viruses of 6 of 79 dogs sampled in New York City in March-April 1973 was obtained through the use of four different methods for measurement of anti-hemagglutination and anti-neuramini dase antibody.
Although many animals are susceptible to experimental infection with influenza viruses, natural infection has been documented in only a limited number of species [1], In view of current speculation that animals may serve as a reservoir for pandemic variants of influenza A viruses [2,3], evidence of natural infection in domestic animals with human influenza viruses has con siderable epidemiologic importance. Experimental infection with both influ enza A and B viruses has been produced in domestic dogs (reviewed by N ikitin et al. [4]). However, little evidence has been provided for natural in fection of this species. N ikitin et al. [4] sampled sera from 271 domestic dogs following H3N2 influenza epidemics in the United States and United King dom, and demonstrated antibody to A/Hong Kong/6/68 virus in 5.9% of animals. Reports from the Soviet Union (cited in Easterday and C ouch [1]) noted that canine illnesses and death in the Vladivostok area increased in assoAddress inquiries to: Dr. E. D. K ilbourne, Department of Microbiology, Mount Sinai School of Medicine, Fifth Avenue and 100th Street, New York, N Y 10029 (USA)
Downloaded by: HSC Library-Serials Dept., SUNY 129.49.5.35 - 11/28/2017 3:35:54 PM
Received: February 16, 1976.
316
K ilbourne/K ehoe
dation with influenza epidemics in man, in February 1970 and December 1971, and that Hong-Kong-Iike influenza viruses were isolated from dogs dur ing those periods. However, definitive serologic evidence for infection with a specific influenza virus rests on demonstration of antibody to both hemag glutinin and neuraminidase antigens, particularly in the case of animal influ enza A viruses in which hemagglutinin and neuraminidase appear in varying combinations [5], Serologic tests used in previous studies [4], ‘hemadsorptionneutralization’ and complement fixation, do not distinguish between anti body to hemagglutinin and to neuraminidase and probably are mediated principally through the hemagglutinin antigen. Therefore, it seems appro priate to add further evidence to that already reported on infection of do mestic dogs with influenza. In the present study, we have employed serologic techniques capable of independent assessment of both anti-hemagglutinin and anti-neuraminidase responses. Single serum specimens from 79 dogs in the New York metropolitan area were obtained randomly from clinic visits during March and April of 1973. Animals were unselected and their history with respect to possible influenza exposure was not known. Anti-hemagglutinin antibody was measured in receptor-destroying-enzyme (RDE)-treated sera by conventional hemag glutination inhibition (HI) tests as previously described [6], employing A/ England/42/72 and A/Aichi/68 (H3N2) influenza viruses. Sera with which inhibitory activity was demonstrated were also titrated in plaque neutraliza tion [plaque inhibition (PI)] tests [7] against recombinant virus A/England/42/ 72-Prague/l/56 (H3i:ngNeql) to measure antibody specific for the hemag glutinin. As noted in table I. positive HI tests against one or the other antigen were observed in 10 of 79 dogs studied, with demonstration of higher titers against the 1968 virus in all but one instance. Nonspecific serum inhibitors have often obfuscated interpretation of HI tests in surveys of animal sera for influenza antibody [8]; however, neutralization is effected only by antibody except in the case of certain inhibitor-sensitive *+ ’ H2N2 strains [9], There fore, it was important to show that of the 10 sera positive by HI, 7 had signifi cant levels of anti-H3 neutralizing (PI) antibody. Further evidence for infection of the animals with H3N2 virus was pro vided by demonstration of anti-neuraminidase (anti-N2) antibody in the sera of 6 dogs by specific inhibition of viral neuraminidase (Nl [6]) (table I). In 2 of these dogs, neuraminidase antibody was demonstrated also by plaque-size reduction (PSR) [7] of the recombinant virus H0pn8N2j3ng that possesses only the N2 antigen of H3N2 virus and hence is a neuraminidase-specific serologic reagent. This virus was also employed in NI tests.
317
Influenza in Dogs
Table /. Demonstration in canine sera of antibodies reactive with hemagglutinin and neu raminidase antigens of influenza A virus of H3N2 subtype Dog
Anti-hemagglutinin antibody hemagglutination inhibition H3N2(Eng/72) H3N2(Aichi/68)
20a 24a 2 II 22 25 26a 3 32 20
20" 10 20 10 20 160 it8N 2Eng
PSRC H0pBsN2Bng
400 400 400 800 6,400
< 400 < 400 < 400
