(~) INSTITUT PASTEUR/ELsEVIER
Res. Viral. 1992, 143, 199-204
Paris 1992
Natural infection of dogs by influenza C virus J.C. Manuguerra (') and C. H a n n o u n Unitd d'Ecologie Virale, Institut Pasteur, 75724 Paris Cedex 15
SUMMARY To date, only one seroepidemiological survey, carried out in Japan, gave a strong indication that dogs may be naturally infected by the influenza C virus, long considered to be exclusively human. In the present work, 134 serum samples were collected during the winter of 1988/89 from dogs aged 6 months to 16 years in northern France. Samples were tested for the presence of antibodies to influenza C virus by both haemagglutination inhibition (Hll and ELISA. Using antibody absorption by staphylococcal protein A, we demonstrated the specificity of the results. In 62 % of cases, the results were identical using the t w o methods. Significant HI activity was found in 32 % of the 134 tested sera and titres ranged from 20 to 320. Of the sera tested, 42 % were positive by ELISA and titres ranged from 500 to 8,000. The discordant results are discussed. The population tested was divided into five age groups : < 4 years, 4 to 6 years, 7 to 9 years, 10 to 11 years and > 12 years. The distribution of antibodies in the tested canine population, in contrast to that of humans, did not show a significant degree of association with age.
Key-words: Dog, Influenza C virus; Antibodies, Natural infection, Northern France. INTRODUCTION
Natural infection of dogs by influenza viruses was first established by a seroepidemiological survey of a canine population carried out in the United States and Great Britain in 1968 during the Hang Kong pandemic (Nikithin, 1972). In 1975, haemadsorption-inhibiting antibodies directed against A/Hang Kong/1/68 (H3N2) were found in 10 out of 79 dog sera (Kilbourne and Kehoe, 1975). Another seroepizootiological survey in dogs (Madhawan and Agarwal, 1976) suggested that A/England/4/72 (H3N2)- and AJCaen/72 (H3N2)-like viruses infected dogs after having circulated in human population. Furthermore, Romvary et aL isolated influenza A/Budapest/4/72-1ike viruses from dogs in Hungary. From these studies, it is strongly suggested that influenza A subtype (H3N2) viruses (*) Corresponding author.
can naturally infect dogs, but the rate of seropositives among them is too low to consider dogs as a potential reservoir for influenza A/H3N2 viruses. Influenza C virus was considered as exclusively human until it was isolated from pigs in China (Yuanji et al., 1983). Although pig isolates appeared to be very close to human viruses, they proved to differ in genome analysis (Yuanji and Desselberger, 1984). In Japan, the distribution of antibodies against influenza B and C viruses was studied in 2656 sera from different species of mammals (horses, cattle, pigs, dogs, cats, minks and rats) as well as in 740 avian sera by the haemagglutination inhibition (HI) test. For influenca C virus, the rate of seropositives was 6.6 °70 (Kawano et al., 1978). In China (Yuanji et al., 1983), it
200
J.C. M A N U G U E R R A A N D C. H A N N O U N
was shown that experimental infection of pigs was possible and transmission from pig to pig was demonstrated. In Japan, it was shown ( H o m m a , 1986) that 148 dog sera a m o n g 471 (31.4 %) were positive for influenza C virus. That group infected mongrel dogs with the C / A n n A r b o r / l / 5 0 strain o f influenza C virus. A clinical, serological and virological follow-up was then carried out on the infected dogs. After the initial infection, all animals became ill: the symptoms were nasal discharge, swelling eyelids with conjunctivitis and m u c o u s epiphora. S y m p t o m s lasted up to 10 days for most o f the dogs. Clinical features o f the experimental disease in dogs were identical to those naturally observed in man. Dogs were resubmitted to exposure to virus after 50 and 100 days. Like man, they were reinfected with virus shedding and disease. It is noteworthy that, in dogs, symp t o m s were more severe after experimental infection with influenza C virus than after e x p e r i m e n t a l i n f e c t i o n with A / P h i l a d e l phia/33/58 and B/Jersey/163/62 (Todd and Cohen, 1968). In other m a m m a l species, like mice, hamsters, monkeys and rats, the infection leads to seroconversion but not to disease. Dogs may represent a special target for influenza C virus, c o m p a r e d to other types o f influenza viruses. Since there has been only one seroepidemiological survey to date that suggests natural infection o f dogs by influenza C virus ( H o m m a , 1986), we sought to confirm this, and to provide possible epidemiological implications for man.
MATERIALS AND METHODS D o g sera
Dog sera were collected in the winter of 1988/1989 at the "Ecole Nationale V6t~rinaire d'Alfort" and were provided to us by Prof. J.-L. Pouchelon. Most of the 134 dogs originated from the lie-de-France (region of Paris). All ages were represented in this study. A high proportion of animals were 9-12 years
INF-C = influenzaC. PTS = PBS+Tween+ serum. RDE = receptor-destroying enzyme.
old, while a smaller number were between 3 to 9 years old. The mean age was 7.8 years. The youngest dog was 6-months old and the oldest 16-years old. Dogs less than one year old were grouped together. This study included 30 breeds, while 25 % of the population consisted of mongrels. Animals were brought in for examination for various reasons. Only one dog was vetted for respiratory disorder. Blood was collected for medical purposes.
Serum treatments for HA and HI tests
Removal of non-specific inhibitors was performed with neuraminidase from Vibrio cholerae (strain 4Z), usually referred to as RDE (receptor-destroying enzyme). It was provided by the WHO Collaborating Center for Influenza, Centers for Disease Control, Atlanta (USA). Treatment of dog sera for nonspecific inhibitors was performed as follows: a solution of RDE of V. cholerae (100 U/ml) was added at 4/1 (v/v) and incubated at 37°C overnight. A sodium citrate solution was then added (2.5 % ; w/v) and sera were heated for 30 min at 56°C; 2 vol. of PBS p H 7 . 2 (NaCI, 100mM; KC1, 5 . 5 m M ; Na2HPO 4, 20 mM; KH2PO4, 3 mM) were finally added to the mixture. Final serum dilution was 1/10. After treatment, natural agglutinins to chicken erythrocytes were removed by a 3-h incubation at 4°C with a 50 % suspension (v/v) of chick erythrocytes under constant shaking.
Haemagglutination (HA) and haemagglutination inhibition (HI) tests
The C/Johannesburg/1/66(C/JHB/1/66) strain of influenza C (INF-C) virus, provided by Dr. Herrlet (Germany) and selected for its relatively good growth capacity in chicken embryo, was used throughout these tests. Virus was grown in the allantoic cavity of 8-day old embryonated eggs at 32°C and fluids were harvested 3 days after inoculation. Fresh INF-C-infected allantoic fluid was diluted in PBS to obtain a standard viral concentration of 4 HA units. The tests were performed at 4°C to prevent elution, using U-shaped microplates as described previously (Dowdle et al., 1979). The results were read after 1 h. The titre was the reciprocal value of the last dilution where agglutination or its inhibition was clearly visible. The detection of antibody was considered as positive when the HI titre was at least 20.
RT TE
= room temperature. = Tris-HCI/EDTA.
NATURAL
201
I N F E C T I O N O F D O G S B Y I N F L U E N Z A C VIRUS
Virus purification
INF-C-infected fluid (C/JHB/1/66), which was produced as described above, was clarified at 3,000 rpm for 30 rain. Supernatant was then centrifuged at 25,000 g for 150 rain in an angular rotor (Beckman J14). Pellets were resuspended in 0.1 ml Tris-HCI-EDTA (TE) buffer and left at 4°C overnight ; 0.3 ml of TE was added for disintegration of pelleted particles by sonication (30 s, 100 W, 20 Hz). The homogenized suspension was loaded on a continuous potassium-tartrate gradient (10 to 50 °70 w/v) in TE buffer and centrifuged at 200,000 g for 300 min in a swinging bucket rotor (Kontron TST41). The purified viral suspension was dialysed against PBS overnight (4°C) and used for experiments. The protein content was determined by Bradford's method, using bovine serum albumin as protein standard, with the Bio-Rad protein assay (Bradford, 1976). Stock virus contained 516 ~.g of protein/ml.
ELISA procedure The plates were coated with a purified virus suspension (10 ~.g/ml) in carbonate-bicarbonate buffer pH = 9.6 overnight at 4°C. Each step of the procedure was followed by 5 plate washings with PT (PBS + 0.1 070 Tween 20; Merck, Germany). After saturation by a 30-min contact with PTS (PT + 1 % foetal calf serum) and washing, the samples (200 g.l) diluted in PTS were dispensed and incubated for 2 h at room temperature (RT). After a second saturation by a 30-min contact with PTS, peroxidase-labelled antibodies directed against the heavy and light chains of dog IgG (Biosys, France) (1/4,000 in PTS) were placed in contact for 2 h at RT. Then, 200 I.d of ortho-phenylenediamine 3.7 mM in phosphate-citric acid buffer pH 6, containing 10 IA H202 (130 volumes) were added to the wells. After 15 min, the reaction was stopped by addition of 0.5 N citric acid. Absorbances were read at 450 rim. On each plate, 12 wells were left blank. Absorbance values were corrected by deducing the mean value of the blank wells. In order to prevent interference with egg proteins, the sera were diluted in PT complemented with 10 % noninfected allantoic fluid. The titre was the reciprocal value of the last dilution scored as positive. The limit of positivity was based upon sera (1) that were devoid of HI activity and (2) that corresponded to the lowest optical densities (OD from 0.100 to 0.170) after correction, obtained by ELISA for the first dilution (1/500). Their global mean was m I and their mean standard error SEM i. They could be considered as true negatives and were taken as negative controls, in the absence of absolute negative or positive canine standards. The limit of non-negativity for
ELISA was set at I = m l + 4 SEMt. In the experiments presented here, I varied from 0.12 to 0.40. Serum treatment with protein A/sepharose
Treated sera (200 I.d) were incubated with 50 ~.g of microbeads of protein A/sepharose extracted from Staphylococcus aureus (LKB-Pharmacia, Sweden) for l h under shaking (Richman et al., 1982). The reaction was stopped by centrifugation, removal of the supernatant sera and transfer to other vials.
RESULTS A relatively high p e r c e n t a g e o f d o g sera tested by E L I S A and H I c o n t a i n e d a n t i b o d i e s against i n f l u e n z a C virus
The 134 dog sera were examined by two different methods. As shown in table I, the rates of seropositives for influenza C virus in the tested population were 32 and 42 °70 in HI and ELISA, respectively. The n u m b e r o f sera positive by at least one technique was 75, i.e. 56 070. From these data, it is possible to evaluate the sensitivity o f the tests : 57 070 for HI (43 × 100/75) and 76 070 for E L I S A (56 x 100/75).
Table I. Detection of antibody to influenza C virus
by EL1SA and HI. ELISA Negative Positive
Total
Negative 59 (44 %)
32 (23.8 %)
91 (67.9 °/0)
HI
Positive
Total
19 24 (14. l °70) (17.9 070)
43 (32 070)
78 56 (58.2 070) (41.8 070)
134
A total of 134 serum samples was tested by ELISA and HI for antibody to influenza C virus. For each technique, the number and the percentageof positive and negativeserumsamples(I34 on the whole) are shown.
202
J.C. M A N U G U E R R A A N D C. H A N N O U N
WUI~Lql OP S E ~
Fig. 1. Distribution of antibodies directed against influenza C virus in the dog population. The titres are expressed by the last positive dilution; 0 corresponds to negative result. For HI test, l, 2, 3, 4, and 5 represent 20, 40, 80, 160 and 320, respectively. For ELISA, 1, 2, 3, 4 and 5 are for 500, 1,000, 2,000, 4,000 and 8,000.
As shown in figure I, the highest HI titre was 320 for 1 out of the 134 sera from the tested population. About 4.5 °70 of the tested sera (6 out of 134) showed high HI titres (>t 80). As shown in figure 1, the highest ELISA titre was 8,000 for 1 out of the 134 sera. No more than 3.7 070 of the tested sera showed high ELISA titres (>/ 4,000). In 62 070 of cases, the results were identical in both tests (HI + and ELISA ÷ or H I - and E L I S A - ) , but 32 sera (24 070) were H I - and E L I S A + while 19 (14 070) were HI + and E L I S A - . Sixteen sera demonstrated high ELISA titres though negative in HI tests. Only two sera showed high HI titres though they were negative in ELISA. In order to increase the specificity of ELISA, we decided to set the limit of "non-negativity" at l = m 1 + 4 S E M t i n s t e a d o f t h e usual
m z+ 2 SEM r The specificity of the results was also investigated by antibody absorption using protein A/sepharose. Protein A (extracted and purified from S. aureus) is known to have good binding capacity to canine IgG (Richman et al., 1982). After absorption with protein A, all sera became negative by both methods. This clearly indicated that the serum c o m p o u n d s responsible for HI activity and adsorption on coated plates were removed by protein A, suggesting that these c o m p o u n d s were antibodies rather than non-specific inhibitors.
Distribution of antibodies against influenza C virus in the tested population showed no significant degree of association with age The dog population was divided into 5 age groups : 0 to 3 years of age, 4 to 6 years old, 7
N A T U R A L INFECTION OF DOGS B Y INFLUENZA C VIRUS
to 9, 10 to 11 and 12 to 16. Each group contained from 25 to 29 animals. The indices of seropositives by age group varied from 32 to 58 °7o. The highest proportion of seropositives was found in adults aged 7 to 9 years, and the lowest proportion of seroposittves was in the 0-3 yearold group. Chi-square analysis between the age groups defined above and the proportion of seropositives as determined by ELISA did not show any significant degree of association with age: ;~2=5.8, alpha > 0.05. A similar result was obtained by chi-square analysis between the age groups and the proportion of seropositives, as determined by HI tests.
DISCUSSION
Strain C / J H B / 1 / 6 6 of influenza C virus, though isolated in 1966, was chosen for use in the present study because the genomic and consequently, the antigenic variability undergone by this virus is low and non-cumulative with time (Buonaugurio et al., 1985, 1986); moreover, its growth capacity in eggs is suitable. Purified virus was used to coat plates for ELISA. Though there was no evidence of major egg protein contamination, as checked by Western blotting (data not shown), non-infected allantoic fluid was added to the dilution of sera as previously done for human sera by Troisi and Monto (1981). Overall results indicate a significant association between the two tests. This is consistent with the report of Troisi and Monto (1981) concerning human sera. As expected, the sensitivity of ELISA was slightly higher than that of the HI test. Antibodies detected by ELISA, which were devoid of HI activity, were probably directed against internal viral proteins not involved in haemagglutination, such as the M or NP proteins, or else were in amounts too small to inhibit haemagglutination. As discussed by Troisi and Monto (1981) for human sera, this may have contributed to the greater sensitivity of ELISA, which could have been even higher if we had decided to lower the limit of "non-negativity" to m I + 2SEMi; however, the aim of our study
203
was to confirm the presence of antibodies in dogs, and we preferred lower sensitivity rather than the obtaining of non-specific results. The limit and the first dilution (1/500) may also account for the 19 dog sera which were unexpectedly positive by HI but negative by ELISA. According to our recent study in man (Manuguerra et ai., 1992), since every doublepositive serum was confirmed by Western blotting, we feel it is valid to consider a serum as truly positive for INF-C if is positive in both ELISA and HI. In that same study, we suggested that the confirmation of double-negative results has no influence upon epidemiological conclusions, since they are almost always confirmed. In the case of discordant results by ELISA and HI in human sera, confirmation by Western-blotting is likely if HI or ELISA titres are high. In the present study, the index of seropositives for influenza C virus varies from 32 to 42 °/0, which is close to the initial findings reported in Japan (Homma, 1986). The index of seropositives, despite variations, remains relatively high for all age groups. This study confirms that influenza C virus infection does indeed occur and is, in fact, common in dogs, as once described in Japan. Since the virus has not been isolated from these dogs, however, it is not possible to be sure that the antibodies correspond to a human agent: there could eventually be a closely related agent specific for dogs, or cross-reacting antibodies induced by a hypothetical related antigen. These possibilities are unlikely, and experimental evidence has shown that human influenza C virus can infect dogs with clinical manifestations similar to those observed in humans, and can induce antibody production (Ohwada et al., 1986). Moreover, in experimentally infected animals, virus shedding lasts several weeks: since the rate of seropositive dogs in the tested population is relatively high (32 to 42 o70), the role played by this species in the general epidemiology of influenza C virus must be elucidated to determine if virus exchanges are frequent between the two species.
204
J.C. M A N U G U E R R A
Acknowledgements We wish to thank J.-L. Poucheion, Professor at the Ecole Nationale V~t6rinaire d'Alfort, for providing us with samples of dog serum, and Claudine Rousseaux and Franqoise Ventre for their excellent technical assistance.
Infection naturelle du chien par le virus grippal C J u s q u ' ~ p r e s e n t seule une enqu~te s6ro~pid~miologique r6alis6e au J a p o n portant sur la pr6valence des anticorps dirig6s contre le virus de la grippe de type C chez le chien sugg6rait fortement que cet animal pouvait ~tre naturellement infect6 par cet agent consid6r6 Iongtemps c o m m e exclusivement humain. Un total de 134 s6rums provenant de chiens •~g6s de 6 mois ~ 16 ans vivant dans la moiti6 nord de la France a ~t~ collect6 durant l'hiver 1988/1989. La pr6sence d'anticorps contre la grippe C dans ces ~chantillons a 6t~ d~tect~e par les techniques d'inhibition de l'h6magglutination (IH) et ELISA. P a r i'usage de prot~ine A staphylococcique, en plus des contr61es habituels pour chacun des tests employ6s, nous avons d6montr6 qu'il s'agissait bien d'anticorps et non d'inhibiteurs non sp6cifiques. Dans 62 % des cas, ies r~sultats ~taient positifs par les deux m~thodes. Des titres IH significatifs ont ~t6 trouv6s pour 32 % des 134 s6rums test~s et s'6tageaient de 20 320, alors que 42 % ont &6 trouv6s positifs par ELISA, les titres ailant de 500 ~ 8.000. Les r~sultats discordants sont discut6s. La population canine soumise ~ enqu~te a ~t6 divis~e en 5 groupes d'~ges: < 4arts, 4 b. 6 arts, 7 ~ 9 ans, 10 et I I ans et > 12 ans. Contrairement /~ ce qui est d6crit chez l ' h o m m e , la distribution des anticorps dans cette population ne semble pas li6e ~ i'~.ge.
Mots-cl~s: Chien, Virus grippal C; Anticorps, Infection naturelle, Nord de la France.
References Bradford, M. (1976), A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye-binding. Analyt. Biochem., 72, 248. Buonaugurio, D.A., Nakada, S., Fitch, W.M. & Palese,
A N D C. H A N N O U N P. (1986), Epidemioiogy of influenza C virus in man : multiple evolutionary lineages and low rate of change. Virology, 153, 12-21. Buonaugurio, D.A., Nakada, S., Desselberger, U., Kristal, M. & Palese, P. 0985), Noncumulative sequence changes in the hemagglutinin genes of influenza C virus isolates. Virology, 146, 221-232. Dowdle, W.A., Kendal, A.P. & Noble, G.R. (1979), Influenza viruses, in "Diagnostic procedures for viral, rickettsial and chlamydial infections" (5th) (E. Lennette and N. Schmidt) (pp. 585-606). American Society for Microbiology, Washington DC. Homma, M. (1986), Epidemioiogical characteristics of type C influenza, in "Option for the control of influenza" (pp. 125-138). Alan R. Liss, New York. Kawano, J., Onta, T., Kida, H. & Yanagawa, R. (1978), Distribution of antibodies in animals against influenza B and C viruses. Jap. J. Vet. Res., 26, 74-80. Kilbourne, E.D. & Kehoe, J.M. (1975), Demonstration of antibodies to both haemagglutin and neuraminidase antigens of H3N2 influenza A virus in domestic dogs. lntervirology, 6, 315-318. Madhawan, H.N. & Agarwal, S.S. (1976), Seroepidemiology of human and canine influenza in Pondichery South India during 1971-1974. Indian J. Med. Res., 64, 835-840. Manuguerra, J.-C., Hannoun, C. & Aymard, M. (1992), Influenza C virus infection in France. J. Infect., 24, 91-99. Nikithin, T. (1972), Epidemiological studies of A/HongKong/68 virus infection in dogs. Bull. Org. mond. Sant~, 47, 471-479. Ohwada, K., Kitame, F. & Homma, M. (1986), Experimental infection of dogs with type C influenza virus. Microbiol. Immunol., 30, 451-460. Richman, D.D., Cleveland, P.H., Oxman, M.N. & Johnson, K.M. (1982), The binding of staphylococcal protein A by the different animal species. J. Immunol., 128, 2300-2305. Romvary, J., Rozsa, J. & Farkas, E. (1975), Infection of digs and cats with the Hong-Kong influenza A(H3N2) virus during an epidemic period in Hungary. Acta Veterinaria, 25, 255-259. Todd, J.D. & Cohen, D. (1968), Studies of influenza in dogs. - - I. Susceptibility of dogs to natural and experimental infection with human A2 and B strains of influenza virus. Amer. J. Epidemiol., 87, 426-438. Troisi, C.L. & Monto, A.S. (1981), Comparison of enzymelinked immunosorbent assay and hemagglutination inhibition in a seroepidemiological study of influenza type C infection. J. clin. MicrobioL, 14, 516-521. Yuanji, G. & Desselberger, U. (1984), Genome analysis of influenza C virus isolated in 1981-82 from pigs in China. J. gen. Virol., 65, 1857-1872. Yuanji, G., Fengen, J., Ping, W., Min, W. & Jiming, Z. (1983), Isolation of influenza C virus from pigs and experimental infection of pigs with influenza C virus. J. gen. ViroL, 64, 177-182.
Res. Viral. 1992, 143, 199-204
Paris 1992
Natural infection of dogs by influenza C virus J.C. Manuguerra (') and C. H a n n o u n Unitd d'Ecologie Virale, Institut Pasteur, 75724 Paris Cedex 15
SUMMARY To date, only one seroepidemiological survey, carried out in Japan, gave a strong indication that dogs may be naturally infected by the influenza C virus, long considered to be exclusively human. In the present work, 134 serum samples were collected during the winter of 1988/89 from dogs aged 6 months to 16 years in northern France. Samples were tested for the presence of antibodies to influenza C virus by both haemagglutination inhibition (Hll and ELISA. Using antibody absorption by staphylococcal protein A, we demonstrated the specificity of the results. In 62 % of cases, the results were identical using the t w o methods. Significant HI activity was found in 32 % of the 134 tested sera and titres ranged from 20 to 320. Of the sera tested, 42 % were positive by ELISA and titres ranged from 500 to 8,000. The discordant results are discussed. The population tested was divided into five age groups : < 4 years, 4 to 6 years, 7 to 9 years, 10 to 11 years and > 12 years. The distribution of antibodies in the tested canine population, in contrast to that of humans, did not show a significant degree of association with age.
Key-words: Dog, Influenza C virus; Antibodies, Natural infection, Northern France. INTRODUCTION
Natural infection of dogs by influenza viruses was first established by a seroepidemiological survey of a canine population carried out in the United States and Great Britain in 1968 during the Hang Kong pandemic (Nikithin, 1972). In 1975, haemadsorption-inhibiting antibodies directed against A/Hang Kong/1/68 (H3N2) were found in 10 out of 79 dog sera (Kilbourne and Kehoe, 1975). Another seroepizootiological survey in dogs (Madhawan and Agarwal, 1976) suggested that A/England/4/72 (H3N2)- and AJCaen/72 (H3N2)-like viruses infected dogs after having circulated in human population. Furthermore, Romvary et aL isolated influenza A/Budapest/4/72-1ike viruses from dogs in Hungary. From these studies, it is strongly suggested that influenza A subtype (H3N2) viruses (*) Corresponding author.
can naturally infect dogs, but the rate of seropositives among them is too low to consider dogs as a potential reservoir for influenza A/H3N2 viruses. Influenza C virus was considered as exclusively human until it was isolated from pigs in China (Yuanji et al., 1983). Although pig isolates appeared to be very close to human viruses, they proved to differ in genome analysis (Yuanji and Desselberger, 1984). In Japan, the distribution of antibodies against influenza B and C viruses was studied in 2656 sera from different species of mammals (horses, cattle, pigs, dogs, cats, minks and rats) as well as in 740 avian sera by the haemagglutination inhibition (HI) test. For influenca C virus, the rate of seropositives was 6.6 °70 (Kawano et al., 1978). In China (Yuanji et al., 1983), it
200
J.C. M A N U G U E R R A A N D C. H A N N O U N
was shown that experimental infection of pigs was possible and transmission from pig to pig was demonstrated. In Japan, it was shown ( H o m m a , 1986) that 148 dog sera a m o n g 471 (31.4 %) were positive for influenza C virus. That group infected mongrel dogs with the C / A n n A r b o r / l / 5 0 strain o f influenza C virus. A clinical, serological and virological follow-up was then carried out on the infected dogs. After the initial infection, all animals became ill: the symptoms were nasal discharge, swelling eyelids with conjunctivitis and m u c o u s epiphora. S y m p t o m s lasted up to 10 days for most o f the dogs. Clinical features o f the experimental disease in dogs were identical to those naturally observed in man. Dogs were resubmitted to exposure to virus after 50 and 100 days. Like man, they were reinfected with virus shedding and disease. It is noteworthy that, in dogs, symp t o m s were more severe after experimental infection with influenza C virus than after e x p e r i m e n t a l i n f e c t i o n with A / P h i l a d e l phia/33/58 and B/Jersey/163/62 (Todd and Cohen, 1968). In other m a m m a l species, like mice, hamsters, monkeys and rats, the infection leads to seroconversion but not to disease. Dogs may represent a special target for influenza C virus, c o m p a r e d to other types o f influenza viruses. Since there has been only one seroepidemiological survey to date that suggests natural infection o f dogs by influenza C virus ( H o m m a , 1986), we sought to confirm this, and to provide possible epidemiological implications for man.
MATERIALS AND METHODS D o g sera
Dog sera were collected in the winter of 1988/1989 at the "Ecole Nationale V6t~rinaire d'Alfort" and were provided to us by Prof. J.-L. Pouchelon. Most of the 134 dogs originated from the lie-de-France (region of Paris). All ages were represented in this study. A high proportion of animals were 9-12 years
INF-C = influenzaC. PTS = PBS+Tween+ serum. RDE = receptor-destroying enzyme.
old, while a smaller number were between 3 to 9 years old. The mean age was 7.8 years. The youngest dog was 6-months old and the oldest 16-years old. Dogs less than one year old were grouped together. This study included 30 breeds, while 25 % of the population consisted of mongrels. Animals were brought in for examination for various reasons. Only one dog was vetted for respiratory disorder. Blood was collected for medical purposes.
Serum treatments for HA and HI tests
Removal of non-specific inhibitors was performed with neuraminidase from Vibrio cholerae (strain 4Z), usually referred to as RDE (receptor-destroying enzyme). It was provided by the WHO Collaborating Center for Influenza, Centers for Disease Control, Atlanta (USA). Treatment of dog sera for nonspecific inhibitors was performed as follows: a solution of RDE of V. cholerae (100 U/ml) was added at 4/1 (v/v) and incubated at 37°C overnight. A sodium citrate solution was then added (2.5 % ; w/v) and sera were heated for 30 min at 56°C; 2 vol. of PBS p H 7 . 2 (NaCI, 100mM; KC1, 5 . 5 m M ; Na2HPO 4, 20 mM; KH2PO4, 3 mM) were finally added to the mixture. Final serum dilution was 1/10. After treatment, natural agglutinins to chicken erythrocytes were removed by a 3-h incubation at 4°C with a 50 % suspension (v/v) of chick erythrocytes under constant shaking.
Haemagglutination (HA) and haemagglutination inhibition (HI) tests
The C/Johannesburg/1/66(C/JHB/1/66) strain of influenza C (INF-C) virus, provided by Dr. Herrlet (Germany) and selected for its relatively good growth capacity in chicken embryo, was used throughout these tests. Virus was grown in the allantoic cavity of 8-day old embryonated eggs at 32°C and fluids were harvested 3 days after inoculation. Fresh INF-C-infected allantoic fluid was diluted in PBS to obtain a standard viral concentration of 4 HA units. The tests were performed at 4°C to prevent elution, using U-shaped microplates as described previously (Dowdle et al., 1979). The results were read after 1 h. The titre was the reciprocal value of the last dilution where agglutination or its inhibition was clearly visible. The detection of antibody was considered as positive when the HI titre was at least 20.
RT TE
= room temperature. = Tris-HCI/EDTA.
NATURAL
201
I N F E C T I O N O F D O G S B Y I N F L U E N Z A C VIRUS
Virus purification
INF-C-infected fluid (C/JHB/1/66), which was produced as described above, was clarified at 3,000 rpm for 30 rain. Supernatant was then centrifuged at 25,000 g for 150 rain in an angular rotor (Beckman J14). Pellets were resuspended in 0.1 ml Tris-HCI-EDTA (TE) buffer and left at 4°C overnight ; 0.3 ml of TE was added for disintegration of pelleted particles by sonication (30 s, 100 W, 20 Hz). The homogenized suspension was loaded on a continuous potassium-tartrate gradient (10 to 50 °70 w/v) in TE buffer and centrifuged at 200,000 g for 300 min in a swinging bucket rotor (Kontron TST41). The purified viral suspension was dialysed against PBS overnight (4°C) and used for experiments. The protein content was determined by Bradford's method, using bovine serum albumin as protein standard, with the Bio-Rad protein assay (Bradford, 1976). Stock virus contained 516 ~.g of protein/ml.
ELISA procedure The plates were coated with a purified virus suspension (10 ~.g/ml) in carbonate-bicarbonate buffer pH = 9.6 overnight at 4°C. Each step of the procedure was followed by 5 plate washings with PT (PBS + 0.1 070 Tween 20; Merck, Germany). After saturation by a 30-min contact with PTS (PT + 1 % foetal calf serum) and washing, the samples (200 g.l) diluted in PTS were dispensed and incubated for 2 h at room temperature (RT). After a second saturation by a 30-min contact with PTS, peroxidase-labelled antibodies directed against the heavy and light chains of dog IgG (Biosys, France) (1/4,000 in PTS) were placed in contact for 2 h at RT. Then, 200 I.d of ortho-phenylenediamine 3.7 mM in phosphate-citric acid buffer pH 6, containing 10 IA H202 (130 volumes) were added to the wells. After 15 min, the reaction was stopped by addition of 0.5 N citric acid. Absorbances were read at 450 rim. On each plate, 12 wells were left blank. Absorbance values were corrected by deducing the mean value of the blank wells. In order to prevent interference with egg proteins, the sera were diluted in PT complemented with 10 % noninfected allantoic fluid. The titre was the reciprocal value of the last dilution scored as positive. The limit of positivity was based upon sera (1) that were devoid of HI activity and (2) that corresponded to the lowest optical densities (OD from 0.100 to 0.170) after correction, obtained by ELISA for the first dilution (1/500). Their global mean was m I and their mean standard error SEM i. They could be considered as true negatives and were taken as negative controls, in the absence of absolute negative or positive canine standards. The limit of non-negativity for
ELISA was set at I = m l + 4 SEMt. In the experiments presented here, I varied from 0.12 to 0.40. Serum treatment with protein A/sepharose
Treated sera (200 I.d) were incubated with 50 ~.g of microbeads of protein A/sepharose extracted from Staphylococcus aureus (LKB-Pharmacia, Sweden) for l h under shaking (Richman et al., 1982). The reaction was stopped by centrifugation, removal of the supernatant sera and transfer to other vials.
RESULTS A relatively high p e r c e n t a g e o f d o g sera tested by E L I S A and H I c o n t a i n e d a n t i b o d i e s against i n f l u e n z a C virus
The 134 dog sera were examined by two different methods. As shown in table I, the rates of seropositives for influenza C virus in the tested population were 32 and 42 °70 in HI and ELISA, respectively. The n u m b e r o f sera positive by at least one technique was 75, i.e. 56 070. From these data, it is possible to evaluate the sensitivity o f the tests : 57 070 for HI (43 × 100/75) and 76 070 for E L I S A (56 x 100/75).
Table I. Detection of antibody to influenza C virus
by EL1SA and HI. ELISA Negative Positive
Total
Negative 59 (44 %)
32 (23.8 %)
91 (67.9 °/0)
HI
Positive
Total
19 24 (14. l °70) (17.9 070)
43 (32 070)
78 56 (58.2 070) (41.8 070)
134
A total of 134 serum samples was tested by ELISA and HI for antibody to influenza C virus. For each technique, the number and the percentageof positive and negativeserumsamples(I34 on the whole) are shown.
202
J.C. M A N U G U E R R A A N D C. H A N N O U N
WUI~Lql OP S E ~
Fig. 1. Distribution of antibodies directed against influenza C virus in the dog population. The titres are expressed by the last positive dilution; 0 corresponds to negative result. For HI test, l, 2, 3, 4, and 5 represent 20, 40, 80, 160 and 320, respectively. For ELISA, 1, 2, 3, 4 and 5 are for 500, 1,000, 2,000, 4,000 and 8,000.
As shown in figure I, the highest HI titre was 320 for 1 out of the 134 sera from the tested population. About 4.5 °70 of the tested sera (6 out of 134) showed high HI titres (>t 80). As shown in figure 1, the highest ELISA titre was 8,000 for 1 out of the 134 sera. No more than 3.7 070 of the tested sera showed high ELISA titres (>/ 4,000). In 62 070 of cases, the results were identical in both tests (HI + and ELISA ÷ or H I - and E L I S A - ) , but 32 sera (24 070) were H I - and E L I S A + while 19 (14 070) were HI + and E L I S A - . Sixteen sera demonstrated high ELISA titres though negative in HI tests. Only two sera showed high HI titres though they were negative in ELISA. In order to increase the specificity of ELISA, we decided to set the limit of "non-negativity" at l = m 1 + 4 S E M t i n s t e a d o f t h e usual
m z+ 2 SEM r The specificity of the results was also investigated by antibody absorption using protein A/sepharose. Protein A (extracted and purified from S. aureus) is known to have good binding capacity to canine IgG (Richman et al., 1982). After absorption with protein A, all sera became negative by both methods. This clearly indicated that the serum c o m p o u n d s responsible for HI activity and adsorption on coated plates were removed by protein A, suggesting that these c o m p o u n d s were antibodies rather than non-specific inhibitors.
Distribution of antibodies against influenza C virus in the tested population showed no significant degree of association with age The dog population was divided into 5 age groups : 0 to 3 years of age, 4 to 6 years old, 7
N A T U R A L INFECTION OF DOGS B Y INFLUENZA C VIRUS
to 9, 10 to 11 and 12 to 16. Each group contained from 25 to 29 animals. The indices of seropositives by age group varied from 32 to 58 °7o. The highest proportion of seropositives was found in adults aged 7 to 9 years, and the lowest proportion of seroposittves was in the 0-3 yearold group. Chi-square analysis between the age groups defined above and the proportion of seropositives as determined by ELISA did not show any significant degree of association with age: ;~2=5.8, alpha > 0.05. A similar result was obtained by chi-square analysis between the age groups and the proportion of seropositives, as determined by HI tests.
DISCUSSION
Strain C / J H B / 1 / 6 6 of influenza C virus, though isolated in 1966, was chosen for use in the present study because the genomic and consequently, the antigenic variability undergone by this virus is low and non-cumulative with time (Buonaugurio et al., 1985, 1986); moreover, its growth capacity in eggs is suitable. Purified virus was used to coat plates for ELISA. Though there was no evidence of major egg protein contamination, as checked by Western blotting (data not shown), non-infected allantoic fluid was added to the dilution of sera as previously done for human sera by Troisi and Monto (1981). Overall results indicate a significant association between the two tests. This is consistent with the report of Troisi and Monto (1981) concerning human sera. As expected, the sensitivity of ELISA was slightly higher than that of the HI test. Antibodies detected by ELISA, which were devoid of HI activity, were probably directed against internal viral proteins not involved in haemagglutination, such as the M or NP proteins, or else were in amounts too small to inhibit haemagglutination. As discussed by Troisi and Monto (1981) for human sera, this may have contributed to the greater sensitivity of ELISA, which could have been even higher if we had decided to lower the limit of "non-negativity" to m I + 2SEMi; however, the aim of our study
203
was to confirm the presence of antibodies in dogs, and we preferred lower sensitivity rather than the obtaining of non-specific results. The limit and the first dilution (1/500) may also account for the 19 dog sera which were unexpectedly positive by HI but negative by ELISA. According to our recent study in man (Manuguerra et ai., 1992), since every doublepositive serum was confirmed by Western blotting, we feel it is valid to consider a serum as truly positive for INF-C if is positive in both ELISA and HI. In that same study, we suggested that the confirmation of double-negative results has no influence upon epidemiological conclusions, since they are almost always confirmed. In the case of discordant results by ELISA and HI in human sera, confirmation by Western-blotting is likely if HI or ELISA titres are high. In the present study, the index of seropositives for influenza C virus varies from 32 to 42 °/0, which is close to the initial findings reported in Japan (Homma, 1986). The index of seropositives, despite variations, remains relatively high for all age groups. This study confirms that influenza C virus infection does indeed occur and is, in fact, common in dogs, as once described in Japan. Since the virus has not been isolated from these dogs, however, it is not possible to be sure that the antibodies correspond to a human agent: there could eventually be a closely related agent specific for dogs, or cross-reacting antibodies induced by a hypothetical related antigen. These possibilities are unlikely, and experimental evidence has shown that human influenza C virus can infect dogs with clinical manifestations similar to those observed in humans, and can induce antibody production (Ohwada et al., 1986). Moreover, in experimentally infected animals, virus shedding lasts several weeks: since the rate of seropositive dogs in the tested population is relatively high (32 to 42 o70), the role played by this species in the general epidemiology of influenza C virus must be elucidated to determine if virus exchanges are frequent between the two species.
204
J.C. M A N U G U E R R A
Acknowledgements We wish to thank J.-L. Poucheion, Professor at the Ecole Nationale V~t6rinaire d'Alfort, for providing us with samples of dog serum, and Claudine Rousseaux and Franqoise Ventre for their excellent technical assistance.
Infection naturelle du chien par le virus grippal C J u s q u ' ~ p r e s e n t seule une enqu~te s6ro~pid~miologique r6alis6e au J a p o n portant sur la pr6valence des anticorps dirig6s contre le virus de la grippe de type C chez le chien sugg6rait fortement que cet animal pouvait ~tre naturellement infect6 par cet agent consid6r6 Iongtemps c o m m e exclusivement humain. Un total de 134 s6rums provenant de chiens •~g6s de 6 mois ~ 16 ans vivant dans la moiti6 nord de la France a ~t~ collect6 durant l'hiver 1988/1989. La pr6sence d'anticorps contre la grippe C dans ces ~chantillons a 6t~ d~tect~e par les techniques d'inhibition de l'h6magglutination (IH) et ELISA. P a r i'usage de prot~ine A staphylococcique, en plus des contr61es habituels pour chacun des tests employ6s, nous avons d6montr6 qu'il s'agissait bien d'anticorps et non d'inhibiteurs non sp6cifiques. Dans 62 % des cas, ies r~sultats ~taient positifs par les deux m~thodes. Des titres IH significatifs ont ~t6 trouv6s pour 32 % des 134 s6rums test~s et s'6tageaient de 20 320, alors que 42 % ont &6 trouv6s positifs par ELISA, les titres ailant de 500 ~ 8.000. Les r~sultats discordants sont discut6s. La population canine soumise ~ enqu~te a ~t6 divis~e en 5 groupes d'~ges: < 4arts, 4 b. 6 arts, 7 ~ 9 ans, 10 et I I ans et > 12 ans. Contrairement /~ ce qui est d6crit chez l ' h o m m e , la distribution des anticorps dans cette population ne semble pas li6e ~ i'~.ge.
Mots-cl~s: Chien, Virus grippal C; Anticorps, Infection naturelle, Nord de la France.
References Bradford, M. (1976), A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye-binding. Analyt. Biochem., 72, 248. Buonaugurio, D.A., Nakada, S., Fitch, W.M. & Palese,
A N D C. H A N N O U N P. (1986), Epidemioiogy of influenza C virus in man : multiple evolutionary lineages and low rate of change. Virology, 153, 12-21. Buonaugurio, D.A., Nakada, S., Desselberger, U., Kristal, M. & Palese, P. 0985), Noncumulative sequence changes in the hemagglutinin genes of influenza C virus isolates. Virology, 146, 221-232. Dowdle, W.A., Kendal, A.P. & Noble, G.R. (1979), Influenza viruses, in "Diagnostic procedures for viral, rickettsial and chlamydial infections" (5th) (E. Lennette and N. Schmidt) (pp. 585-606). American Society for Microbiology, Washington DC. Homma, M. (1986), Epidemioiogical characteristics of type C influenza, in "Option for the control of influenza" (pp. 125-138). Alan R. Liss, New York. Kawano, J., Onta, T., Kida, H. & Yanagawa, R. (1978), Distribution of antibodies in animals against influenza B and C viruses. Jap. J. Vet. Res., 26, 74-80. Kilbourne, E.D. & Kehoe, J.M. (1975), Demonstration of antibodies to both haemagglutin and neuraminidase antigens of H3N2 influenza A virus in domestic dogs. lntervirology, 6, 315-318. Madhawan, H.N. & Agarwal, S.S. (1976), Seroepidemiology of human and canine influenza in Pondichery South India during 1971-1974. Indian J. Med. Res., 64, 835-840. Manuguerra, J.-C., Hannoun, C. & Aymard, M. (1992), Influenza C virus infection in France. J. Infect., 24, 91-99. Nikithin, T. (1972), Epidemiological studies of A/HongKong/68 virus infection in dogs. Bull. Org. mond. Sant~, 47, 471-479. Ohwada, K., Kitame, F. & Homma, M. (1986), Experimental infection of dogs with type C influenza virus. Microbiol. Immunol., 30, 451-460. Richman, D.D., Cleveland, P.H., Oxman, M.N. & Johnson, K.M. (1982), The binding of staphylococcal protein A by the different animal species. J. Immunol., 128, 2300-2305. Romvary, J., Rozsa, J. & Farkas, E. (1975), Infection of digs and cats with the Hong-Kong influenza A(H3N2) virus during an epidemic period in Hungary. Acta Veterinaria, 25, 255-259. Todd, J.D. & Cohen, D. (1968), Studies of influenza in dogs. - - I. Susceptibility of dogs to natural and experimental infection with human A2 and B strains of influenza virus. Amer. J. Epidemiol., 87, 426-438. Troisi, C.L. & Monto, A.S. (1981), Comparison of enzymelinked immunosorbent assay and hemagglutination inhibition in a seroepidemiological study of influenza type C infection. J. clin. MicrobioL, 14, 516-521. Yuanji, G. & Desselberger, U. (1984), Genome analysis of influenza C virus isolated in 1981-82 from pigs in China. J. gen. Virol., 65, 1857-1872. Yuanji, G., Fengen, J., Ping, W., Min, W. & Jiming, Z. (1983), Isolation of influenza C virus from pigs and experimental infection of pigs with influenza C virus. J. gen. ViroL, 64, 177-182.
