Jap. J. M. Sc. & Biol., 16, 65-81, 1963 STUDIES ON A COMMON ANTIGEN BETWEEN A1 AND A2 INFLUENZA VIRUSES FUMIO NISHIKAWA Department of Bacteriology I, National Institute of Health, Tokyo (Received: January 30th, 1963) In the early stage of the Asian influenza pandemic of 1957, several virus strains were isolated from patients among the inhabitants in Singapore and among the American marines of the Far East bases. Hemagglutination-inhibition (HI) test for influenza virus showed that these strains did not share a common antigen with any other type A or B influenza virus which had been isolated before 1957. However, in sera of the pati- ents significant antibody rises in complement-fixation (CF) test were observed when type A soluble (S) antigen was used (Meyer et al., 1957). Afterwards, laboratory tests for diagnosis of Asian influenza were carried out as a rule throughout the world using Asian strain as antigen for HI test or S antigen of type A for CF test. In 1958, Asian influenza virus was designated A2 sub-group of influenza type A virus by the Expert Committee on Respiratory Virus Diseases (Expert Committee on Respiratory Virus Diseases, 1959). Later, Choppin et al. (1958) reported the existence of a minor common antigen between A2 virus and other type A viruses, which was discovered by HI test . They also observed that a few patients infected with A2 influenza were found to have a positive antibody response in HI test not only to A2 but also to Swine influenza or FM1 strain. Jensen et al. (1958) also reported that in a few patients infected with A2 influenza rises of antibody against A1 or A were proved in HI test or in CF test by virus (V) antigen. At the same time they reported that some patients did not show any antibody response to A2 but, in a certain grade, positive response to some other type A virus. Boger et al. (1957) observed that a few individuals among a group of people who received a polyvalent influenza vaccine which did not contain A2 strain showed antibody rises even to A2. On the contrary, Hilleman et al . (1958) reported a few positive cases even to A1 or Swine virus among a group of people who received an A2 monovalent vaccine. At the outbreaks of influenza epidemic of 1960 in Japan , the author obtained similar results as mentioned above that, in testing some paired sera collected from patients, rises of antibody were proved against both A1 and A2 by HI test . However, all viruses isolated from these patients were found to belong to A2 , and no A1 virus was isolated at all. From the foregoing, it seemed to be one of the most important problems to clarify whether or not a common antigen really exists between A1 and A2 viruses , although sera of patients indicated antibody rises to both antigens as mentioned above . The present paper describes the results of an antigenic analysis of A1 and A2 西 川 文雄(国 立 予防 衛 生研 究 所 細 菌 第一 部) 65 66 NISHIKA WA Vol. 16 influenza viruses carried out in our laboratory using HI test and neutralization test in eggs. From these results it can safely be said that these viruses of two sub-groups may possess a minor common antigen as expected. In addition to the results, the cases which were positive to A 1 and A 2 in HI test during the epidemic of influenza from 1957 to 1962 in Japan are presented and discussed. MATERIALS AND METHODS Virus strains : Nine strains of A 1, 15 strains of A 2 and 4 strains of type B influenza virus were used. All these strains except A 2Singapore/1/57 A 2and A 2/Japan/307/57 were isolated in our laboratory at the time of influenza outbreaks. The allantoic fluid harvested from infected eggs with each of these strains was used as an antigen for HI test. Human sera : Unless special mention is made, the human sera employed were those collected from influenza patients by prefectural health laboratories in Japan and sent to our laboratory for serological diagnosis. Antisera : Rooster and rabbit antisera were prepared by the following procedures ; A rooster was given simultaneously with 2 cc of infected allantoic fluid (having a hemagglutination titer of higher than 1 : 256) by the intravenous route and with 10 cc of the same material by the intra- peritoneal route for immunization. These injections were repeated four times at 3 or 4 day intervals. A rabbit was also injected by the same procedure with an exception of 5 cc intravenously given instead of 2 cc, and these immunizations were carried out seven or eight times at 3 or 4 day intervals. Animals were bled a week after the last injection. HI test : HI test was carried out by the procedure routinely used in our laboratory ; For pretreatment of sera, one volume of a human serum was mixed with 3 volumes of RDE solution* and incubated overnight, then heated for 30 minutes at 56•Ž. The sera thus treated were serially diluted by two-fold steps, and the diluted sera were respectively added with 4 hemagglutination units of virus and 0.5% suspension of chicken red cells. Sedimentation patterns were read after standing for one and a half hours at 4 to 20•Ž. In the cases of HI tests for rooster and rabbit antisera, different procedures for the pretreatment were used, which will be indicated later. Neutralization test in eggs: The pretreated rooster antisera were diluted with ordinary broth by two-fold dilution, and each of diluted sera was mixed with an equal volume of properly diluted virus. Then the mixtures were kept at 37•Ž for 30 minutes and inoculated intra-allantoically into four eggs per dilution. After 48 hours, the eggs were chilled and examined for hemagglutination. Neutralizing titers of sera were calculated as 50% neutralizing end-point. Vaccines : Vaccines used in this experiment were kindly supplied from the Society of the Biological Products. RESULTS Elimination of Non-specific Inhibitors in Normal Rooster and Rabbit Sera In the case of HI test for influenza virus, it is important, first of all, to eliminate * Concentration of RDE solution was determined as follows ; Two sets of serial two-fold dilution were made from Vibrio cholerae culture filtrate (RDE) with ordinary broth, and an equal volume of normal rabbit serum was added to each tube of the both dilution series. One more set of serial two-fold dilution was made from the same sample of RDE with ordinary broth and one- third volume of human serum whose antibody titers was less than 1 : 16 was added to each tube of the dilution. The mixtures were heated at 56•Ž for 30 minutes, after an overnight incubation at 37•Ž. HI test for each set of dilution series was carried out with three different antigens, the first two mixed with normal rabbit serum being against heated Lee strain and against living egg- line A 1/Kojiya/1/52 strain, respectively, and the remaining one mixed with human serum against living egg-line A 2/Adachi/2/57 strain. The highest initial dilution of RDE that showed complete hemagglutination to any of these three antigens was used as the concentration of RDE for treat- ment of human sera. 1963 A1 AND A2 INFLUENZA VIRUSES 67 the non-specific inhibitors from sera to be tested, because sera of many species of animals including human beings have been found to contain the inhibitors in various grades. In this respect various procedures had been attempted by many workers so far. Among them RDE and periodate-treatments seemed to be the most useful for elimina- tion of the inhibitors with little loss of antibodies. (Tyrrel & Horsfall, (1952), Sampaio, (1952), Expert Committee on Respiratory Virus Diseases (1959), Sugiura (1959), Sasaki (1959), Harboe et at. (1960), Shiratori et at. (1961) ). According to their results, however, RDE-treatment seemed to be effective in rooster sera, while in rabbit sera these two treatments were not always effective. Therefore, as preliminary experiments, the following procedures for elimination of the inhibitors in normal rooster and rabbit sera were investigated ; In the case of rooster sera, one volume of serum was treated with 3 volumes of RDE solution as in the case of treatment of human sera. As shown in Table 1, non-specific inhibitors were found to be completely eliminated from 4 Table 1. Elimination of non-specific inhibitors in normal rooster sera. * Sera of No . 2, 3, 4 and 8 were treated with a four-times concentrated RDE solution. samples tested. In another experiment, one volume of serum was mixed with an equal volume of four-time concentrated RDE solution. Results were also the same for 4 samples as shown in Table 1. Both the procedures were used for elimination of the inhibitors in rooster antisera in the following experiments. In the case of rabbit sera it was found that the elimination of the inhibitors by RDE-treatment alone was not enough against A2. A complete elimination of the inhibitors from rabbit sera was successful in our laboratory by means of RDE-treatment in addition to NaIO4-treatment. The procedure was as follows ; One part of serum was at first mixed with 1.5 parts of 1/40 M NaIO4, and kept at 37•Ž for 30 minutes, then added with a half part of 1 % glycerin. Thus the treated serum was again added with 1 part of RDE solution. The RDE-treatment was the same as in the case of 68 NISHIKAWA Vol.