In the Early Stage of the Asian Influenza Pandemic of 1957, Several Virus Strains Were Isolated from Patients Among the Inhabita

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 patients 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 treatment 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 elimination 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. 16 1963 A1 AND A2 INFLUENZA VIRUSES 69

Fig. 1. Cross-HI test among A1 and A2 influenza virus strains with rooster antisera (1). human sera. As shown in Table 2, the non-specific inhibitors to A1 and A2 were completely eliminated from 11 samples tested.

Cross-HI Test between A1 and A2 Influenza Viruses Cross-HI test on rooster antisera against 8 strains of A1 and 9 strains of A2 which were pretreated with RDE were carried out against A1 antigens of 8 strains and A2 antigens of 11 strains. As illustrated in Figs. 1 and 2, five A1 antisera have been found to react against antigens of some A2 strains, and six A2 antisera reacted against antigens of some A1 strains. Similar results were obtained in cross-HI test using the pretreated rabbit antisera. Each three antisera of A1 and A2 were tested against A1 antigen of 7 strains and A2 antigen of 8 strains. As shown in Table 3, all three A1 antisera were found for 70 NISHIKA WA Vol. 16

Fig. 2. Cross-HI test among A1 and A2 influenza virus strains with rooster antisera (2). react against one or more A2 antigens, and two A2 antisera were found to show HI against one or more A1 antigens though in a low titer. However, in these results observed from the rooster and rabbit antisera, a uniformity of cross-reactivity among antigens and antisera of strains used was not always obtained. In other words, antisera of those A1 strains which were proved to be inhibited by antisera of some A2 strains did not always inhibit the latter strains, and vice versa. Moreover, the coincidence of cross-reactivity between rooster and rabbit antisera were not always obtained. From these results, although the titer of HI antibody against heterologous sub-group of antigen was shown to be much lower when compared with the homologous sub- 1963 A1 AND A2 INFLUENZA VIRUSES 71 group of antigen, it can safely be said that there is a minor common antigen in HI test between A 1 and A2 influenza viruses.

Cross-Neutralization Test in Eggs between A1 and A2 Influenza Viruses Cross-neutralization tests in eggs were made with 2 rooster antisera of A1 and 3 of A2 versus 4 of A1 and 7 of A2 viruses. As shown in Table 4, all 5 antisera tested have been found to neutralize one or more strains of the heterologous sub-group in a low dilution. However, in these tests, the cross-reactivity between the strains of A1 and A2 did not seem to coincide with that obtained in HI test.

Table 4. Cross-neutralization test in eggs among A1 and A2 influenza virus strains with rooster antisera.

* Titer calculated was less than 2. ** Not tested.

HI Test on Sera of Patients Infected with A2 Influenza Virus From the above findings, special interest was concentrated on to clarify whether or not sera of patients infected with A2 influenza virus really react not only against A2 but also against A1. The paired sera were collected from patients of the Asian influenza epidemic in 1957 up to 1962, and tested for this purpose. In 1957 to 1958: In Japan, the Asian influenza pandemic began in the middle of May of 1957, and prevailed until the end of April of 1958. A large number of cases were reported throughout Japan, and 378 patients were serologically proved in our laboratory as A2 influenza infection. As shown in Table 5, among them 4 cases of paired sera were found to react against A1 antigen as well as A 2, and 1 case did not react against A2 but was found to do against A1. All these sera came from people of 8 to 16 years of age. In 1959: In the spring of 1959, an influenza-like respiratory disease prevailed in Shibata Camp of the Japanese Ground Self-Defence Forces. Throat washings and paired sera of the patients were collected for tests. All attempts at virus isolation from the materials tested were unsuccessful. However, 6 of 17 cases gave a four-fold or greater antibody increase to A1 influenza virus, 8 of them to A2 and the remaining 3 to both 72 NISHIKA WA Vol. 16

Table 5. Positive sera against heterologous antigen (A1) in the HI test from patients of the Asian influenza pandemic in 1957.

* Antibody titers in first/second sample . ** First sample taken/second sample taken .

Table 6. HI tests on sera of patients in Shibata Camp of Japanese Ground Self-Defense Forces in the spring of 1959.

* Antibody titers in acute/covalescent serum . sub-groups. Although this epidemic was evidenced later to be caused by infection of A2 influenza virus, the question of why some of these patients showed antibody rise to A1 alone or both of A1 and A2 remained unsolved at that time of epidemic. In 1960: In an influenza epidemic occurring during the season from winter to early spring in 1960, paired sera of 104 patients were proved positive in antibody rise on HI test in our laboratory. Among them 78 cases showed antibody rise to A2, 4 1963 A1 AND A2 INFLUENZA VIRUSES 73

Table 7. Positive sera against heterologous antigen (A1) in the HI test from the A2 influenza epidemic in 1960.

* Antibody titers in first/second sample . * * First sample taken/second sample taken . # Isolation was done with materials pooled with two to five specimens from each local group.

cases to A1 and 22 cases to both A1 and A2. Viruses isolated at this epidemic were found to belong to A2, but no A1 virus was isolated at all. Of 26 patients who showed significant antibody rises to A1 or both A1 and A2, 18 individuals were under 13 years of age, 4 were 14 tca 30 years, and 4 were over 30 years. (Table 7). Moreover, five of these patients were found to come from Daito-nishi primary school, in Saitama prefecture. Therefore, the following sero-epidemiological surveys were made in this respect. That is, after the epidemic season was over, school children in two primary schools, Daito-nishi and Daito-higashi primary school, were bled. In the former, 74 NISHIKAWA Vol. 16 about 50 children of each 4th and 5th grade were bled, and in the latter, about 100 children of 4th grade were bled for test. In the former, the epidemic was dominant in 5th grade and less in 4th, and in the latter, no epidemic was observed. As shown in Fig. 3, distributions of antibody titers against A1 were almost the same between the 4th and 5th grade of Daito-nishi primary school in spite of the different incidence. That is, modes of the distribution were about 1:64 in both the grades. On the other hand, antibody distribution against A1 of children in 4th grade of Daito-higashi primary school was remarkably different from both grades of Daito-nishi primary school, that is, distribution of antibody titers was deviated to 1:16 or less. But in all the groups, antibody distributions against A2 and type B viruses were almost in the same ranges.

Fig. 3. HI antibody distribution among school children in Daito-nishi and Daito-higashi primary schools after the epidemic of influenza in 1960.

Although the question of why the HI titer to A2 of children in Daito-higashi primary school was as high as that in Daito-nishi primary school remained unsolved, these findings indicated that A1 antibodies of school children in Daito-nishi primary school might have been reacted due to A2 infection through the epidemic. In 1961 to 1962: No epidemic of A2 influenza except for sporadic cases was reported in 1961 in Japan, but type B influenza prevailed throughout the country. In 1962, a wide-spread epidemic of A2 influenza broke out. About one hundred children in Okazu primary school in Yokohama were bled in order to investigate the f ulctuation of antibody in some population through the epidemic. Bleedings were conducted twice at the interval of one year, that is, in March of 1961 when type B influenza epidemic was over, and in March of 1962 when type A2 influenza ceased. 1963 A1 AND A2 INFLUENZA VIRUSES 75

Distribution of antibody titers of these children in 1961 and 1962 are shown gra- phically in Fig. 4. Before summarizing these data, those of the individuals who had no serological evidence of infection with A2 influenza or received polyvalent influenza vaccine were excluded in advance to avoid a possible confusion. As shown in Fig. 4, in second samples, distribution of antibody titers to A2 moved markedly to a range of a high titer. Distribution of antibody titers to A1 had also a similar tendency although it was less remarkable comparing with A2. On the other hand, that to type B was unchanged or rather had a tendency to move to a low titer.

•¬ Bled in March, 1961 •¬Bled in March, 1962

Fig. 4. HI antibody distribution of serologically positive cases of influenza among school children at Okazu primary school in 1962.

For analysing distribution of antibody titers among children who did not develop

any manifestation of clinical infection during the epidemic, a questionnaire was given

to each individual on whether it experienced the disease or not. Seventeen children

stated really no experience of the disease, yet sera of 11 among them showed antibody

rises. Distribution of titers of these 11 sera is shown in Fig. 5. As shown in this

figure, it was similar as that shown in Fig. 4. This means that the grade of antibody

response after inapparent infection of influenza did not differ from that of apparent

infection, and this result is in good agreement with what was investigated at Daito-

nishi primary school in 1960.

HI Antibody Rise to A1 in Sera of Individuals Vaccinated

with A2 Monovalent Vaccine

The effect of A 2 monovalent vaccine on HI antibody rise to A1 was investigated.

At an asylum for the aged in Kanagawa prefecture, 0.1 cc of calcium phosphate 76 NISHIKAWA Vol. 16

•¬ Bled in March. 1961 •¬Bled in March , 1962

Fig. 5. HI antibody dstribution among children with inapparent infection of influenza at Okazu primary school in 1962.

Table 8. Positive sera against heterologous antigen (A1) in HI test from persons vaccinated with A2 monovalent influenza vaccines

precipitated vaccine containing 1,000 CCA/cc of A2/Adachi/2/57 strain was injected subcutaneously to 69 persons over 60 years old, and 0.1 cc of oil (Bayol F) vaccine containing 500 CCA/cc of the same strain was injected subcutaneously to 150 persons, of the same age group. Sera were collected from each individual three times ; before, one and three months after injection. In most of the cases, HI titer to A2 was remarkably increased after the injection. However, in a few cases the rise of HI titer to A1 was observed accompanying that to A2. As shown in Table 8, in one individual who had received calcium phosphate precipitated vaccine, and 8 individuals whc 1963 A 1 AND A 2 INFLUENZA VIRUSES 77 had received oil vaccine, antibody rise to A 1 was observed as well as to A 2. From the foregoing, it was clarified that injection of A 2 monovalent vaccine into human beings could also produce A 1 antibody response, although the A 1 positive rate was as low as that in the case of natural infection of A 2 influenza.

DISCUSSION

Since the pandemic of the Asian influenza broke out in 1957, many studies on various characteristics of the agent of the disease, A 2 influenza virus, have been reported. Mulder et al. (1958) found HI antibodies to A 2 virus in sera of aged people which were collected before the pandemic, and successively Darvenport (1958) confirmed this finding. This finding eventually led them to the hypothesis that A 2 influenza virus was the cause of pandemic in 1889, and had prevailed until 1918. Nelson et al. (1958) could not find any correlation of HI antibodies to PR 8 or FM 1 with those to A 2, but found a clear correlation of Swine influenza with A 2, when sera collected from the cases of an epidemic in 1957 were tested. Kanemitsu et al. (1962 a) reported that persons who had possessed a high-level antibody against Swine, WS or PR 8 were less susceptible to infection of A 2 influenza virus than those who had antibody in lower levels. On the other hand, attempts on antigenic analysis of influenza viruses by CF test using V antigen were performed by Fabiyi et al. (1958). From their data the author could not help considering that a minor common antigen may exist among strains of A 2 and A 1 or A, although they had not explained about it clearly. Choppin et al. (1958) examined rabbit antisera immunized with A, A 1 or A 2 strains and found that when some strains of A, A 1 or A 2 were used as antigens evidence of slight cross- relationships between A 2 and A 1 or A were observed by HI test. Harboe et al. (1960) also observed a similar finding that some rabbit an.tisera immunized with A 1 strains showed a low HI titers to A 2 antigens and vice versa. However, from their observa- tion, these inhibitions disappeared when the antisera were treated with normal allantoic fluid of the embryonated egg. Their conclusion, therefore, was that there was no evidence of cross-HI between A 2 and other type A strains. As for the contradictory results in HI test mentioned above, the question rises as to whether or not the elimination of non-specific inhibitors in sera tested was proper. Our own preliminary results indicated that the inhibitors in rabbit sera were not always eliminated completely by RDE-treatment, although in the cases of rooster sera this treatment was certainly enough. Therefore, in data presented by Choppin et at., we could not expect a complete destruction of non-specific inhibitors in sera tested. Our attempts to clarify if a common antigen between A 2 and A 1 influenza viruses may be proved in immune rabbit sera after a complete elimination of the inhibitors were performed by means of RDE-treatment with combination of NaI04-treatment. As shown in the present data, this treatment could fulfil the purpose of complete elimination of the inhibitors, and we could confirm the existence of a minor common antigen between A 1 and A 2 in testing both the rooster and rabbit immune sera against A 1 or A 2 virus. Furthermore, these antibodies could not be so eliminated by treatment with normal allantoic fluid as was indicated by Harbo et al.. This means that these antibodies must be considered to be specific, that is, produced by a common antigen of A 1 and A2. 78 NISHIKAWA Vol. 16

The existence of common antigen between A 1 and A 2 was also proved in the

cross-neutralization test in eggs as was shown in the present data . However, as was described before , we could not find the uniformity of cross- reactivity among antigens and antisera of the strains used . This may not mean that the different antigenicity against heterologous sub-group exists among strains , but mean that antigenic capacity of each strain against the heterologous sub-group is so poor that the antibody rise depends mostly upon the susceptibility of the animal that is used for test. As for the common antigen of A 2 and A 1 or A observed in human cases , Choppin et al. (1958) and Nelson et al . (1958) reported separately that rises of HI antibody to Swine, PR 8 or some strains of A 1 were observed in paired sera collected from patients

infected with A 2 influenza in 1957. Henle et al . (1960), Holtermann et al. (1960) also could demonstrated similar findings on human sera in their investigations on the CF test using V antigen.

However, it must be mentioned that the antibody production in naturally infected cases of human influenza differs from that of experimentally immunized animals as

indicated by•gDoctrine of original antigenic sin•h(Hennessy et al . (1955), Davenport et al. (1956) ). Fukumi (1961) reported in this respect that at the time of A 2 influenza epidemic in 1960, the mode of distribution of antibody titers against A 2 virus in con- valescent sera of persons over sixty years of age was markedly higher than that of

younger persons. Kanemitsu et al. (1962 b) also reported that•gDoctrine of original antigenic sin•hcould be applied to analysis of antibody titers against strains belonging to the heterologous sub-group of type A influenza virus in sera of persons who received

natural infection of A 2 influenza or inhalation of A 2 attenuated vaccine . As was shown in our data , in the A 2 influenza epidemic during the period of 1957 to 1962 in Japan, the heterologous antibody rise (Al) among sera of patients infected

with A 2 influenza was specially noticed since 1960 . During this period, the antigenic composition of A 2 viruses seemed to stay unchanged . If it is true, the increase of frequency of heterologous antibody rise may be caused by stimulation of repeated in-

fection of A 2 influenza, which possesses a minor common antigen to A 1 . This may also account for the reason why many of such cases were under 13 years of age as was shown in our data, because individuals of this age group might be infected pri- marily with A 1 influenza. As illustrated in the results obtained from the serological survey at Daito-nishi primary school in 1960, ant at Okazu primary school in 1962, antibody rise in individuals against influenza virus seemed to be almost equivalent between the groups which encountered the same epidemic with apparent and inapparent infections . However, the following questions remained unsolved for further investigation : i) why the distribution of HI titers to A 2 among the individuals in Daito-nishi primary school was as high as that in Daito-higashi primary school where no practical epidemic were reported , ii) why the distribution of antibodies to A 2 in Daito-nishi primary school was not so high comparing with that in Okazu primary school where epidemic of the same type (A 2) occurred in 1962, although the distribution of HI titers to A 1 of the former was higher than that of the latter. In the case of human vaccination with monovalent vaccine , the author could prove that A 2 monovalent vaccine could stimulate A 1 antibody . Similar results have been already reported by Hilleman et al. (1958). However , monovalent or polyvalent vaccine 1963 A1 AND A2 INFLUENZA VIRUSES 79 of sub-groups other than A2 was not investigated in our laboratory upon whether it could produce A 2 antibody, although Boger et al. (1957) has already reported a positive result in contrast to the negative result by Hilleman et al. (1958). As to the antigenic change of influenza virus, one of the explanations is that the antibody level of a population may exert influence on antigenic composition of a prevail- ing virus to produce a new variant. This explanation was supported in serologic surveys of human cases performed year after year, on one hand, and also experimentally proved through serial passages of a virus in its host with antiserum (Archetti & Horsfall, 1950). However, the antigenic change from A 1 to A 2 is so remarkable that it does not seem to be reasonable to interprete this difference with that theory. Therefore, some investigators suggested that A2 virus might have an animal origin and infect the human population by chance (Mulder et al., 1958, Kaplan et al., 1959). On the early works on Asian influenza, it was very doubtful that there existed a common antigen between A1 and A2, but now it was clearly demonstrated, and the explanation will still remain disputable that A2 virus may have its origin from A1 virus even though the antigenic difference between them looks so remarkable.

SUMMARY

The confirmation of the existence of a minor common antigen between A1 and A2 influenza viruses was conducted experimentally with the HI test on immune animal sera and the egg-neutralization test. For the purpose of the elimination of non-specific inhibitors, RDE and NaIO4-treatments were tested. With a complete destruction of the inhibitors in immune sera so treated, it was demonstrated that a minor common antigen existed between A1 and A2 in the HI test. The same was also clearly demonstrated in the egg-neutralization test. In a number of sera from patients infected with A2 influenza virus was also demonstrated antibody rises to both A1 and A2. The heterologous antibody rise (A1) was more pronounced in children under the age of 13 years than in other age groups. Inoculation of an A2 monovalent vaccine also revealed that it could produce a heterologous antibody in a few individuals. The reason of increasing tendency of heterologous antibody rise and the origin of A 2 virus was discussed.

The author is indebted to Dr Hideo Fukumi for his critical supervision and guidance on this report, and to Dr Tadao Sonoguchi who kindly supported our sero-epidemiologic survey in this study. The authors wish to express my deep appreciation to Mr Teij i Sugiyama for his technical assistance in our laboratory.

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