Isolation of Influenza C Virus from Pigs and Experimental Infection of Pigs with Influenza C Virus

J. gen. Virol. (1983), 64, 177-182. Printed in Great Britain 177 Key words: influenza C virus/antibodies/pigs Isolation of Influenza C Virus from Pigs and Experimental Infection of Pigs with Influenza C Virus By GUO YUANJI (KUO YUANCHI), JIN FENGEN, WANG PING, WANG MIN AND ZHU JIMING (CHU CHINMING)* Institute of Virology, Chinese Academy of Medical Sciences, Beifing, China (Accepted 27 August 1982) SUMMARY Fifteen strains of influenza C virus were isolated from abattoir pigs in China in 1981 and antibody against influenza C virus was found in pig sera. Virus survey also showed seasonal activity of influenza C virus in pigs. Additionally, experimental infection of pigs with influenza C virus demonstrated that Chinese domestic pigs could be infected by influenza C virus and that the virus could be transmitted from pig to pig. These results indicate that, like influenza A, influenza C virus can cause natural infection in pigs. To our knowledge, this is the first report of such a finding. INTRODUCTION The 1233 strain of influenza C virus was first isolated from a case of mild respiratory disease (Taylor, 1949). There has been no report of isolation of influenza C virus from animals other than man, nor has influenza C virus been adapted to laboratory animals. More recently, the ecology of influenza viruses has been extensively studied in many countries for more than a decade, and isolation of influenza A viruses from many mammals and birds has been reported. It is generally considered that, in marked contrast to influenza A, influenza B and C viruses have no natural animal host. In the course of studies on the ecology of influenza virus in China, we have isolated influenza C viruses from pigs and detected antibody in pig sera. We also found that influenza C virus could infect pigs and could be transmitted from pig to pig under experimental conditions. METHODS Collection of specimens. Forty pharyngolaryngeal and tracheal swabs were taken twice a month from apparently healthy pigs at a slaughter house in Beijing from January to December 1981. Each specimen consisted of five swabs collected from different pigs, so that forty swabs were pooled to form eight samples. Swabs were placed in tubes containing approximately 5 ml of 50% glycerol in 0-1 M-phosphate-buffered saline ph 7.2 (PBS). One hundred pig serum specimens were collected from pigs every month at the same place. All pigs were raised in Hebei province and Beijing suburbs. Sample treatment and virus isolation. These have been described previously (Guo et al., 1981 a). Briefly, the samples were treated with penicillin, streptomycin, gentamicin and kanamycin at 4 C overnight and were then inoculated into the amniotic and allantoic cavities of 10-day-old chick embryos. The eggs were incubated at 35 C for 72 h and tested for haemagglutinating activity with chick red blood cells at 4 C. Virus passage and preparation of antigen. Ten- to eleven-day-old chick embryos were infected amniotically and allantoically with influenza C/1233/47, C/GL/1167/54, C/Georgia/I/69, C/N J/1/76 or any of the 15 strains of influenza C virus isolated from pigs. A mixture of amniotic and allantoic fluids was used as antigen. All human influenza C virus strains except 1233 were kindly donated by A. P. Kendal, Center for Disease Control, Atlanta, Ga., U.S.A. Strain 1233 was provided by the Virus Type Culture Laboratory of our Institute. Other antigens were allantoic fluids of eggs inoculated with A/PR/8/34 (H 1N1) or B/Lee/40 strains. Preparation of soluble nucleoprotein antigen (NP). NP antigen was prepared according to Robinson & Dowdle (1969). Preparation of immune serum. Strain-specific antisera were prepared in chickens by the conventional method against the following strains: A/swine/Iowa/15/30 (HIN1), A/PR/8/34 (H1N1), A/Tianjing/78/77 (H1N1), A/Zhangjiakou/4/57 (H2N2), A/Guangdong/38/77 (H3N2), B/Lee/40, C/1233/47, C/pig/Beijing/32/81, C/pig/ 0022-1317/83/0000-5304 $02.00 1983 SGM

178 Y.J. GUO AND OTHERS Beijing/818/81 and Newcastle disease virus (NDV). Antisera to parainfluenza types I, II and III were prepared in guinea-pigs. The haemagglutination-inhibition titres of all the above sera against homologous strains were t> 320. Type-specific antisera to influenza A and B viruses prepared by the routine method had complement-fixation titres around 100. Haemagglutination and haemagglutination-inhibition test (HI). These were performed by the routine method, but only chicken red blood cells were used in the test and all tests were done at 4 C. Single radial haemolysis test (SRH). The method has been reported (Guo eta/, 1981 b). In brief, 0-1 ml of washed, packed chicken erythrocytes was mixed with 500 haemagglutinating units of virus in the form of a mixture of amniotic and allantoic fluid and the mixture was allowed to remain at 4 C for 10 rain.the coated cells were then washed once in cold PBS, and packed at 1000 rev/min for 5 min. They were finally made up to a 50~ (v/v) suspension in PBS. The sensitized erythrocytes were then used in preparing haemolysis-in-gel plates according to the procedure described by Schild et al. (1975), except that all testing sera were inactivated at 56 C for 30 rain and were absorbed with 20~ (v/v) packed chicken erythrocytes at 4 C overnight before use. Control plates were prepared similarly, from a mixture of uninfected amniotic and allantoic fluids. Other assays. Neuraminidase was assayed by the standard procedure (Henry et al., 1973). Virus titrations and neutralization tests in eggs were performed according to conventional methods. Counter-immunoelectrophoresis. The method has been described (Influenza Laboratory, Institute of Epidemiology, 1977), and is similar to that reported by Dihz et al. (1976), except that influenza A and B NPs and antibodies to the NPs were used in the test. Experimental infection of pigs. A total of thirteen 50- to 60-day-old pigs of the Chinese Su Bai breed were chosen from a local farm in Beijing. Before experiment, the pigs were kept under observation for several days. Two pigs (group 1) were infected with 1.0 ml of 1 107.5 EIDso of the fourth egg passage material of the C/pig/Beijing/32181 strain by intranasal instillation and kept in No. 1 isolation room. Two pigs (group 2) were infectedwithl.0mlofl x 107.5 EIDsooftheeightheggpassagematerialoftheC/NJ/1/76strainandkeptinNo. 2 isolation room. Two days later, two pigs (group 3) were put in No. 1 isolation room for contact infection with group 1 pigs and two pigs (group 4) were put in No. 2 isolation room for contact infection with group 2 pigs. One control pig was kept in No. 3 isolation room. Twentyzfive days later, two 50-day-old pigs bought from the same farm (group 5) were put into No. 1 isolation room; another two (group 6) were put into No. 2 isolation room for further contact infections. Nasal and pharyngolaryngeal swabs were taken before infection and on days 1 to 14 after infection. Serum was taken from all pigs prior to infection and on days 14, 21, 30 and 50 after infection. The rectal temperature of each pig was recorded daily from 1 day before until 8 days after infection or contact. RESULTS Virus isolation and identification Fifteen isolates with positive haemagglutination against chicken red blood cells were obtained in the form of mixed amniotic and allantoic fluids from eggs infected with pig specimens at the first or second passage. The pig isolates can only be passed in the amniotic sac of chick embryos, but not in the allantoic cavity during the early passages. They were rapidly eluted from chick red blood cells at room temperature and were unable to agglutinate monkey and guinea-pig red blood cells. These properties are similar to those of human influenza C virus (Taylor, 1951). In counter-immunoelectrophoresis, no precipitin line was observed between the NP of these isolates and reference antisera to the NP of influenza A and B viruses. This result indicates that these isolates are neither influenza A nor B viruses. Two positive samples isolated from pigs in January were found to be inhibited by immune serum to C/1233/47 strain in titres greater than or equal to 320, but were not inhibited by other immune sera to influenza A, influenza B, NDV or parainfluenza types. The other isolates were identified with sera to C/1233/47 and C/pig/Beijing/32/81 in the HI test. All these isolates could be inhibited in titres 1> 320. These results indicate that all 15 isolates are probably type C influenza virus. No neuraminidase activity was detected with any of the isolates with haemagglutination titres of 640 to 320. Cross-neutralization tests between C/pig/Beijing/32/81 and C/1233/47 strains were carried out in eggs, using a constant dilution (1:20) of chicken immune serum with HI titre 1280 and various virus dilutions. The control EIDs_o for both C/pig/Beijing/32/81 and C/1233/47 were 10-5.5, but both groups after neutralization were negative at 10-2 dilution, giving neutralization indices over 1000. These results confirm that the isolates are type C influenza virus.

Isolation of influenza C virus from pigs 179 Table 1. Distribution of influenza C virus in pigs and antibody to C/pig/Beijing/32/81 in pig sera collected in different months of 1981 Month of No. of No. of sera Haemolysis diameter collection isolations Name of strain positive* [mean and range (mm)] January 2 C/pig/Beijing/10/81 7 7-9 (5.5-11-0) C/pig/Beijing/32/81 February 3 C/pig/Beijing/107/81 8 10.2 (7.0--12.0) C/pig/Beijing/110/81 C/pig/Beijing/115/81 March 2 C/pig/Beijing/123/81 1 7.5 C/pig/Beijing/128/81 April 1 C/pig/Beijing/294/81 6 7.9 (6.0-10.0) May 4 C/pig/Beijing/368/81 2 7.5 (6.0-9.0) C/pig/Beijing/371/81 C/pig/Beijing/382/81 C/pig/Beijing/383/81 June 0-2 10.0 (7.0-13.0) July 0-1 8.0 August September 0 0 - - 1 2 8.0 9.0 (7.0-11.0) October 0-2 7.0 (6.0-8.0) November 3 C/pig/Beijing/789/81 4 7.3 (6.0-9.0) C/pig/Beijing/815/81 C/pig/Beijing/818/81 December 0-3 7.7 (7.0-8.0) * 100 serum samples were tested each month. The distribution of influenza C viruses isolated from pigs in different months of 1981 is shown in Table 1. All isolates were obtained in the winter and spring seasons, but none was obtained in the summer and autumn seasons. Cross-haemagglutination-inhibition tests between human and pig influenza C viruses Cross-HI tests were performed with two strains of influenza C isolated from pigs in January and December respectively and human influenza C viruses using chicken immune sera. The results are shown in Table 2. It can be seen that cross-reactions were observed among all strains. C/pig/Beijing/32/81, C/pig/Beijing/818/81 and C/NJ/1/76 strains are antigenically similar, but differ slightly from other human strains. Surveys of antibodies to influenza C viruses in pig sera All pig sera were first tested with the C/pig/Beijing/32/81 strain by the HI test; sera with HI titres >I 20 were checked with the same strain by SRH. Sera producing haemolytic zones with diameters of 5 mm or more were considered positive. In spite of HI titres 1> 20, sera with haemolytic zones less than 5 mm were considered negative. The data in Table 1 show that antibody against C/pig/Beijing/32/81 strain was detected every month, in about 3 % (ranging 1 to 8 ~/.) of pig sera. These results further confirm that Chinese domestic pigs are infected with type C influenza viruses in nature. Clinical signs Experimental infection of pigs The rectal temperatures of all pigs were recorded during an 8-day observation period. All temperature measurements were normal, ranging from 37 C to 40.2 C. The other clinical signs, consisting of slight difficulty in breathing and increase in nasal secretion, were observed in group 1 and group 2 pigs inoculated intranasally, but pigs number 1 and 4 recovered very quickly, whereas those in pigs number 2 and 3 lasted 10 days after inoculation. There were no signs of disease in other experimental and control pigs.

180 Y. J. GUO AND OTHERS Table 2. Cross haemagglutination-inhibition between human and pig influenza C viruses* Virus A C/1233/47 C/GL/1167/ C/Georgia/ C/N J/I/76 C/Pig/Beijing/ C/Pig/Beijing/ Serum 54 69 32/81 818/81 C/1233/47 8960 4480 480 800 3840 1600 C/GL/1167/54 800 960 200 200 800 400 C/Georgia/69 50 80 200 80 280 1280 C/N J/1/76 1280 2560 640 3840 6400 8960 C/Pig/Beijing/32/81 2560 2240 1280 2240 8960 8960 C/Pig/Beijing/818/81 3840 3840 1280 4480 7680 * The data presented were confirmed by repeating the test, with similar results. Table 3. Virus excretion in experimental pigs Time after inoculation or contact (days) Pig Pig r x group Treatment given no. 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 ~ 1 Inoculated with 1 - +* + C/Pig/Beijing/32/81 2 + + 2 + + + Inoculated with C/N J/1/76 3 4 + + - + + + 3 In contact with group 1, 5 + +........ 2 days after inoculation 6 + 4 In contact with group 2, 7 + + + t 2 days after inoculation 8 + + 5 In contact with group 1, 9........ 25 days after inoculation 10.... 6 In contact with group 2, 11 25 days after inoculation 12 Control 13............... * '+' indicates positive virus isolation t Death. Measurement of virus excretion from experimental pigs These results are shown in Table 3. It can be seen that virus was isolated from group 1, 2, 3 and 4 pigs on different days after inoculation. It is interesting that the virus can be isolated from pig number 2 in group 1 for at least 9 days after inoculation and from pig number 4 in group 2 for at least 11 days after inoculation. Virus was isolated from group 3 and 4 pigs in contact with the inoculated pigs 2 days after inoculation, but not from group 5 and 6 pigs which were brought into contact 25 days after inoculation. These results indicate that pigs are readily infected experimentally with influenza C viruses isolated from man and pigs and that infection can be transmitted to other pigs. All viruses re-isolated from experimental pigs were identified as influenza C with immune sera prepared against C/pig/Beijing/32/81 and C/NJ/1/76. Antibody response in experimental pigs Antibody was tested by SRH in the sera of inoculated, contact and control pigs. The results are shown in Table 4. The results show that antibody was detected in all sera taken on day 14 or later from those pigs in groups 1, 3 and 5 which were either inoculated with influenza C virus isolated from pigs or had been in contact with the above pigs. In contrast, in groups 2, 4 and 6, which were either inoculated with human influenza C virus or in contact with the above pigs, antibody was detectable only in pigs number 3 and 11. These results might indicate a difference in antibody response between individual pigs to pig and human influenza C viruses. It is interesting that no antibody was detectable in the sera of pigs 4 and 8, in spite of positive virus isolation from them. However, antibody was detected in sera taken from pigs 9 and 10 of

Isolation of influenza C virus from pigs Table 4. Antibody response in experimental pigs Time after inoculation or contact (days) Pig Pig r ~" group Treatment given no. 0 14 21 30 50 Inoculated with 1 0-0" 8-0 8.0 7.0 8.0 1 C/Pig/Beijing/32/81 2 0.0 7-0 6.5 7.0 6.5 2 Inoculated with 3 0.0 7.5 5-5 6.0 NOt C/N J/1/76 4 0.0 0.0 0-0 0.0 0.0 In contact with group 1, 5 0.0 0.0 8.2 7-5 8.0 3 2 days after inoculation 6 0.0 0-0 8.0 8.0 ND In contact with group 2, 7 0.0 ND ND ND ND 4 2 days after inoculation 8 0.0 0.0 0-0 0-0 0-0 In contact with group 1, 9 0.0 0-0 9.0 7.0 ND 5 25 days after inoculation 10 0-0 6.0 9.0 8.0 ND 6 In contact with group 2, 25 days after inoculation 11 12 0-0 0.0 0-0 0-0 5.0 0.0 7-5 0.0 ND ND Control 13 0.0 0.0 0-0 0.0 0.0 * Figures indicate the diameter (mm) of haemolytic zone with undiluted serum. ~" ND, Not done. 181 group 5 and number 11 of group 6, 14 to 30 days after contact with pigs inoculated 25 days previously. This indicates that pigs can remain infectious 25 days after inoculation of pig or human influenza virus. DISCUSSION According to cross-hi and cross-neutralization tests, the pig isolates were identified as influenza C virus. The antigenic structure of the haemagglutinins of C/pig/Beijing/32/81 and C/pig/Beijing/818/81 is very similar and similar to that of C/N J/I/76 strain, but differs slightly from that of other human influenza C virus strains. Kendal (1975) and Nerome et al. 0976) reported that influenza C virus has no neuraminidase. The lack of neuraminidase activity in the pig strains is also consistent with that of human influenza C virus. The lyophilized strain 1233 was obtained from the Virus Type Culture Laboratory of our Institute and was not opened until cross-hi and experimental infection of pigs were carried out in May of 1981, by which time most of the pig strains had already been isolated. An antigenic difference between C/1233 and pig viruses was revealed from the data in Table 2, and confirmed by a repeat test. Furthermore, sensitivity to fl and ~ inhibitors and the capacity to agglutinate rat, mouse and rabbit red blood cells are also different for C/1233 and pig strains (unpublished data). After the first two pig strains, i.e. C/pig/Beijing/10/81 and C/pig/Beijing/32/81 were isolated, successful re-isolation from the original samples was carried out in our laboratory. So, the possibility of laboratory contamination can be ruled out. Serological survey demonstrated that specific antibody against influenza C virus existed in pig sera, further indicating that pigs were infected in nature. We found that the isolation rates were different in different months, indicating that the activity of influenza C virus in pigs was affected by season and may possibly be related to that of influenza C virus in the human population. Virus recovery and antibody rise in experimentally inoculated and contact pigs demonstrated that influenza C viruses can infect pigs and can be transmitted from pig to pig. There were differences in antibody response between pigs inoculated with pig and human influenza C viruses. This may be due to differences in their capacity to multiply in pigs, but this problem needs further investigation. It is very interesting that pigs inoculated with influenza C virus remained infectious for contact pigs for at least 25 days after inoculation, as shown by an antibody rise in pigs 9, 10 and 11. This result is similar to that of an experimental infection of pigs with swine influenza virus (HIN1) reported by Bla]kovi6 et al. (1970) and suggests that influenza C virus might cause persistent infection in pigs. Obviously, further work needs to be done to clarify this point.

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