A PRELIMINARY SEROLOGIC STUDY OF HEPATITIS A VIRUS INFECTION IN JAPAN. YASUO MORITSUGU, TOMOYUKI TANAKA* and TOSHIO SHIKATA*

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1 Japan. J. Med. Sci. Biol., 31, , 1978 A PRELIMINARY SEROLOGIC STUDY OF HEPATITIS A VIRUS INFECTION IN JAPAN YASUO MORITSUGU, TOMOYUKI TANAKA* and TOSHIO SHIKATA* Department of Enteroviruses, National Institute of Health, Gakuen, Musashimurayama, Tokyo ; *Department of Pathology, Wakayama Medical College, Wakayama 610; and **Department of Pathology, Nihon University School of Medicine, Tokyo 173, Japan (Received: January 25, 1978) SUMMARY: Ninety-eight acute non-b hepatitis cases recently observed in Japan and household contacts with these cases were subjected to serologic examinations for hepatitis A; 400 serum specimens obtained in 1971 from healthy individuals living in areas near Tokyo and 16 preparations of human immunoglobulin produced in Japan in 1975 and 1976 were examined for antibody to hepatitis A antigen. Hepatitis A virus infection was confirmed in all 25 patients and in 8 of 26 household contacts found in association with non-b hepatitis outbreaks, and also in 11 of 60 sporadic non-b hepatitis patients, but in none of 13 non-b hepatitis patients found in association with blood transfusion. There was no difference between males and females in the prevalence of antibody to hepatitis A antigen among healthy individuals, however, there was a strong relationship to age. Rates of antibody positives were only 2.5% in the groups younger than 20 years of age, but the rates were markedly higher in the groups older than 30 years of age. An ample amount of antibody to hepatitis A antigen was detected in the preparations of human immunoglobulin. Hepatitis A virus was thus found to be endemic in Japan, but considered not popular during at least these 20 years. Infection with non-a non-b hepatitis virus(es) seems to be common in Japan especially in such cases as sporadic non-b hepatitis or post-transfusion non-b hepatitis. INTRODUCTION The discovery of hepatitis A antigen (HA Ag) and the concurrent demonstration of specific antibody to hepatitis A antigen (anti-ha) in sera of convalescent hepatitis A patients by immune electron microscopy (IEM) by Feinstone, Kapikian and Purcell (1973) brought an important breakthrough in the field of viral hepatitis A. With this technique, the shedding of HA Ag and the subsequent rise of anti-ha were confirmed in experimentally infected cases (Dienstag et al., 1975a; Maynard et al., 1975; Dienstag et al., 1975b) and naturally infected cases (Gravell et al., 1975; Dienstag et al., 1975c). Hepatitis A virus was successfully purified from the livers of infected marmosets (Provost et al., 1975a) and from feces of naturally infected patients and experimentally infected chimpanzees. Such other serologic methods as complement fixation (Provost et al., 1975b), immune adherence hemagglutina- 325

2 326 MORITSUGU et al. Vol. 31 Lion (IAHA) (Miller et al., 1975; Moritsugu et al., 1976) and solid-phase radioimmunoassay (Hollinger et al., 1975; Purcell et al., 1976) have rapidly been developed. Consequently, not only serologic confirmation of individual hepatitis A cases, but also large-scale serologic surveys became feasible by means of such simple, sensitive and specific methods. With HA Ag purified from either the livers of marmosets (Saguinus mystax) experimentally infected with hepatitis A virus or feces obtained from experimentally infected chimpanzees or naturally infected humans, serologic examinations of experimentally infected cases (Krugman, Friedman and Lattimer, 1975), and those from natural outbreaks of hepatitis A (Dienstag et al., 1976a), non-human primate associated outbreaks (Dienstag et al., 1976b), and anti-ha survey (Szmuness et al., 1976; Maynard et al., 1966; Villarejos et al., 1976) were carried out. Since the pioneer study by Okochi and Murakami (1968), the particular importance of hepatitis B virus in Japan has been well documented by many investigators. However, nothing is known about infection with hepatitis A virus in Japan. We engaged in a survey of hepatitis A in Japan for about one year and this communication describes the results of our studies. MATERIALS AND METHODS Serum: Serum samples were obtained from patients diagnosed as acute hepatitis not due to infection with hepatitis B virus. The patients were bled during the acute phase of illness and again during the convalescent phase usually one month or more after the first bleeding. Serum samples were obtained also from the household contacts with some of these patients at the time of the second serum sampling. Serum specimens collected in 1971 from normal individuals living in areas near Tokyo were obtained by random extration from the stock kept at WHO Serum Reference Bank in Tokyo. All serum specimens were stored at -20 C or at -70 C and heated at 56 C for 30 min before examination. Human immunoglobulin preparations: Human immunoglobulin preparations produced in Japan in 1975 and 1976 were obtained from Nichiyaku Co. (Tokyo), Midorijuji Co. (Osaka) and Japan Red Cross Association (Tokyo). Nonspecific IAHA activities found in some of these preparations were removed by centrifugation at 35,000 rpm for 1 hr in 2-ml adapter centrifuge tubes for the Spinco type 40 rotor (Beckman Instruments, Inc., Palo Alto, CA.). After centrifugation, the supernatant fluid was collected and examined for anti-ha. Hepatitis A antigen: The HA Ag preparation used in the present study was obtained from a stool specimen collected early in the illness from a Naval recruit involved in a natural outbreak of hepatitis A in 1974 in San Diego (Hooper et al., 1974; Dienstag et al., 1975c). From this fecel specimen, HA Ag was extracted and partially purified by isopycnic separation in cesium chloride and rate zonal centrifugation in sucrose as described before (Moritsugu

3 1978 HEPATITIS A IN JAPAN 327 et al., 1976). Immune electron microscopy (IEM) : IEM was performed as described by Feinstone et al. (1973). Briefly, 10 ƒêl of the HA Ag preparation, containing about 10 IAHA units of HA Ag, was added to a 2-ml adapter centrifuge tube for the Spinco type 40 rotor which contained 1 ml of 0.01 M Tris (hydroxymethyl) aminomethane buffer (ph 7.5) supplemented with 0.15 M NaCl and 0.5% bovine serum albumin. To the tube, 10 ƒêl of a test serum was then added and thoroughly mixed. After 1-hr incubation at room temperature, the mixture was centrifuged at 23,000 rpm for 90 min. The supernatant fluid was removed; the pellet was resuspended in 25 ƒêl of distilled water. The pellet suspension was then placed on a collodion-coated electron-microscope specimen grid and negatively stained with 3% phosphotungstic acid (ph 7.2). Examination of the grid for HA Ag bound with anti-ha was performed with a JEOL model 100 U electron microscope (Japan Electron Optics Laboratory Co., Tokyo) at a plate magnification of 50,000. The presence of anti-ha in the test serum was rated from 0 to 4 according to the extent of the binding antibody to HA Ag as described by Dienstag, Ailing and Purcell (1976). Immune adherence hemagglutination (IAHA): IAHA assay was performed as described before (Moritsugu et al., 1976). A polyacryl (Lucite) microtiter plate (Nippon Micro-Tec. Co., Tokyo) was used in the present study. Accordingly, instead of the modified diluent to be used in a polystyrene microtiter plate, the diluent fluid described previously by Mayumi et al. (1971), i.e., a veronal buffered saline (VBS), ph 7.5, added with 0.1% gelatin (GVB++), was used. GVB++, GVB++ added with 40 mm ethylenediamine-tetraacetic acid (EDTA-GVB), ph 7.5, guinea pig complement (C'), and dithiothreithol (DTT) were obtained from Eizai Co., Ltd., Tokyo. Human type-o erythrocytes were kindly supplied by Dr. M. Mayumi (Jichi Medical School, Tochigi). A serum sample obtained from chimpanzee No. 753 after infection with hepatitis A virus (Dienstag et al., 1975b) was kindly provided by Dr. R. H. Purcell and used as the control serum with anti-ha. After heating at 56 C for 30 min, serum specimens were first screened for anti-ha at two dilutions, 1:64 and 1:512, then when necessary anti-ha was titrated. For the titration, two sets of serial twofold dilutions of a test serum in GVB++ were prepared in wells of a microtiter plate with microdiluters (Cooke Engineering Co., Alexandria, VA). The wells for anti-ha assay each received a 25-ƒÊ1 amount of the HA Ag preparation diluted so as to contain 4 IAHA units. The other wells received GVB++ and used for the control assay. The plate was incubated at 37 C for 1 hr then the wells each received 25 ƒêl of a C' dilution. The C' dilution giving the highest IAHA differed from one lot to another, but usually in a range from 1:60 to 1:80. After the addition of C', the plate was again incubated at 37 C for 40 min, then 25 ƒêl each of DTT solution (3 mg/ml in EDTA-GVB) and a 1 % suspension of human type-o erythrocytes (suspended in EDTA-GVB) were added and the mixture was well mixed. After 1-hr standing at room temperature, hemagglutination patterns

4 328 MORITSUGU et al. Vol. 31 were rated from 0 to 4 according to the degree of hemagglutination. Hemagglutination of 3 or 4 was considered positive. The antibody titer of a test serum was expressed by the reciprocal of the highest serum dilution giving the specific hemagglutination. The serum sample was considered positive for anti-ha, if the titer was at least eight-fold higher than that of the diluent control. Such sera showing questionable results by IAHA were examined by IEM for the presence of anti-ha. Antibody response to HA Ag was determined usually by simultaneous antibody titrations employing a pair of serum specimens taken during the acute and convalescent phases of illness. In some cases, the antibody response was determined by the examination of a single serum specimen taken in the convalescent phase. A titer of 2,560 or higher, or an eight-fold or greater rise in titer in the convalescent serum was considered to be the response induced by recent infection with hepatitis A virus. RESULTS Serodiagnostic Study Overall results: All of the acute hepatitis cases included in the present study were previously proved not due to infection with hepatitis B virus. Sixty cases of sporadic acute hepatitis, 13 cases of transfusion-associated hepatitis and 25 cases found in association with non-b hepatitis outbreaks were subjected to serologic examinations. In addition to these hepatitis cases, a number of house- TABLE I Hepatitis A cases confirmed by serologic tests hold contacts were also examined. As shown in Table I, the antibody response to HA Ag indicating recent infection with hepatitis A virus was detected in only 11 of the 60 sporadic hepatitis cases and in none of the transfusionassociated cases examined. However, all of the 25 outbreak-associated cases were diagnosed as hepatitis A, and, in addition, in eight of the household contacts, asymptomatic infections with hepatitis A virus were serologically confirmed. Infection with hepatitis A virus was thus confirmed with a total of 44 cases in the present study. Sporadic hepatitis A: As described above, among 60 cases of sporadic acute

5 1978 HEPATITIS A IN JAPAN 329 TABLE II Hepatitis A cases among sporadic non-b hepatitis * anti-ha was examined by IAHA and titers are expresssed in reciprocals of serum dilution as described in MATERIALS AND METHODS. ** Early serum samples were obtained within one month after onset of illness. *** Late serum samples were obtained at an interval of one or more months from the first bleeding for the early serum sample. **** Anti-HA was not examined because no serum sample was available non-b hepatitis, 11 cases were proved to be hepatitis A. No secondary infection was found in any of their household contacts. As shown in Table II, all these cases were adults ranging from 22 to 47 years of age. Ten cases were male and only one was female. All cases except one occurred during the period from December to April. All these patients were Japanese, but very interestingly about half of them apparently acquired the infection during their visits to foreign countries. At the time of infection, i.e., during the period from two to six weeks before onset of illness, five patients were in such countries as India, Nepal, Sri Lanka, Hong Kong or Taiwan. The remaining six patients did not go abroad; accordingly only these patients seemed to have acquired the infection in Japan. Hepatitis A outbreak: Outbreaks of hepatitis A in Japan appeared to be very rare and only two small outbreaks were encountered during the period of the present study. Four cases of hepatitis A occurred in succession in a family in Ohmura in November, The first case was a child who was attending a nursery school. It was retrospectively found that around the time of his illness several cases of jaundice were seen in the nursery, but no serologic confirmation was attempted (M. Yano, personal communication). A small hepatitis A outbreak was observed also in Wakayama in There were a total of 25 hepatitis cases temporarily associated with this outbreak, 21 of which were included in the present study and the diagnoses of all were confirmed. The first case was in June, and other cases continued to occur until September. The index case was a five-year-old boy attending a nursery.

6 330 MORITSUGU et al. Vol. 31 Fig. 1. Anti-HA titers of serum samples taken at the acute phase of illness (black circle) and several months after the outbreak (open circle) from patients and household contacts. Anti-HA was titrated by IAHA as described in MATERIALS AND METHODS. school, and a total of seven cases including this were found in succession among children attending the nursery. All these seven children belonged to different families and the rest of the patients occurred in five of these families. The detail of the outbreak will be reported separately. The results of serologic examinations with 21 patients and 26 household contacts are shown in Fig. 1. A remarkable antibody response to HA Ag was detected in all of the patients examined. In the serum specimens obtained from these cases several months after the end of the outbreak, anti-ha titers were found in a range from 2,560 to 20,480 with a geometric mean titer of 5,939. On the other hand, in the serum specimens collected early within one or two weeks after the onset of illness, anti-ha was found only in low titers. Moreover, the hemagglutination patterns of these serum samples in IAHA experiments were of weak appearance and rated two or three like those observed previously in the serum samples obtained during the early phase of antibody

7 1978 HEPATITIS A IN JAPAN 331 response of an experimentally infected chimpanzee (Moritsugu et al., 1976). In the serum samples obtained from household contacts several months after the end of the outbreak, anti-ha titers were found in a wider distribution. Such higher titers as 5,120 or 10,240 were detected in eight serum samples. These eight contacts were considered to have had asymptomatic infection with hepatitis A virus during the period of the outbreak. Anti-HA was not detected in four household contacts and in the remaining 14 contacts the titers distributed in a range from 80 to 1,280 with a geometric mean titer of 290. Although anti-ha titers in these 14 contacts were quite similar to those found in the sera obtained from patients in the early phase of illness, the hemagglutination patterns in IAHA experiments appeared perfect and generally rated as four throughout the positive range upto one dilution below the endpoint. It was considered that in these 14 persons the antibody was induced by a previous infection. Most of these cases may have been infected with hepatitis A virus probably years before and they were exempted from infection during the present outbreak as they were already immune to hepatitis A. Hepatitis A virus infection of a total of 29 cases was confirmed during the present outbreak. As indicated in Table III, the incidence was equal between male and female, but inversely related to age. Of the 29 cases, 19 TABLE III Hepatitis A cases discovered in association with Non-B hepatitis outbreak TABLE IV Incidence by age and sex of confirmed hepatitis A in Wakayama in 1975 * The numerator indicates the number of hepatitis A cases confirmed and the denominator the number examined.

8 332 MORITSUGU et al. Vol. 31 TABLE V Rate o f hepatitis A infection by age-group among expossed susceptibles in Wakayama in 1975 Total 33 29(88%) * Total exposed persons examined minus 14 immune persons. were children, other eight adults from 20 to 40 years of age, and the other two older than 40 years. The relative rates of infection in these age groups calculated were 86%, 50% and 22%, respectively (Table IV). As described above, the 26 household contacts (Fig. 1) were divided into three groups according to the anti-ha titers of their serum specimens, i.e., eight asymptomatically infected, other 14 probably immune due to prior infection and the other four not infected. All of the eight asymptomatically infected were children. Of the 14 immune, 13 were adults and only one was a child; of the four not infected, two were children and the remaining two adults. When these 14 immune persons were excluded from the exposed population, as indicated in Table V, the rate of hepatitis A incidence appeared to be not different between the young and adult. Therefore, the above-mentioned inverse age-relationship of the relative hepatitis A incidence was probably caused by the age-related immunity against hepatitis A. Sero-epidemiologic Study To confirm the possible age-relation of the immunity to hepatitis A in Japanese population, antibody surveillance was performed. From the stock of serum specimens of WHO Serum Reference Bank in Tokyo collected in 1971 TABLE VI Specification by age, sex and residence o f the donors of serum specimens collected in 1971 in Kanto area

9 1978 HEPATITIS A IN JAPAN 333 TABLE VII Incidence of anti-ha positive by sex among healthy population in Kanto area TABLE VIII Incidence of anti-ha by age-group among healthy persons in Kanto area from healthy individuals living in neighborhood prefectures around Tokyo such as Chiba, Saitama, and Gunma, 400 sera were randomly obtained with the specification of age and sex. As shown in Table VI, of these donors, 163 (40.8%) were urban residents, 150 (37.50) were town residents, and 82 (20.50) were village residents. Anti-HA was found at the equal frequency in males and females (Table VII), but the antibody prevalence strongly related to age (Table VIII). As shown in Table VIII, the rates of antibody positives in the groups younger than 20 years of age were only 2.5%. The rates increased in the groups of ages of twenties and thirties, and thereafter the rates kept a plateau level at about 70 to 75%. When compared by the place of dwelling, the rate of antibody positives appeared to be significantly lower among the population in cities (Table IX). However, when the rates were compared among age-groups, the difference by the place of dwelling was not statistically significant. Human Immunoglobulin Preparation To determine the actual concentrations of anti-ha in human immuno - globulin preparations produced and used in Japan, 16 such preparations produced in 1975 and 1976 were examined by IAHA. As shown in Table X, the

10 334 MORITSUGU et al. Vol. 31 TABLE IX Prevalence of anti-ha by dwelling * The individuals not indicating their dwellings were excluded. ** The numerator indicates the number of anti-ha positives, the denominator the number examined, and the figure in parentheses the percentage of anti-ha positives. TABLE X Anti-HA concentrations o f 16 lots o f human immunoglobulin produced in Japan in 1975 and 1976 Geometric mean titer 3,918 titers of these preparations ranged from 1,024 to 8,192, but the majority (1 preparations) were found to have titers of 4,096. The geometric mean titer calculated was 3,918. DISCUSSION According to the recent report by Szmuness et al. (1976), the rates of antiha positives among people in New York were different by ethnic groups, socioeconomic conditions and ages. The rates were higher in colored population and Chinese-Americans and tended to be lower in people with higher education or high economic standards, but the rates strongly related to age. Similar age or socioeconomic differences of anti-ha positives were observed also in a population in Texas by Maynard et al. (1976). During the outbreak in Wakayama, chances of exposure to hepatitis A virus appeared to be similar between children and adults, but hepatitis A infections were predominantly found among the young population. This inverse age-relation of relative frequency of infection was considered to be due to the higher rate of immunity against hepatitis A among the adult population. The age-related immunity to hepatitis A was indicated also by the antibody survey

11 1978 HEPATITIS A IN JAPAN 335 described in the present report. The age curve of anti-ha positives obtained by the survey consisted of three portions, i.e., the first low-rate phase, the next shift-up phase and the last high-rate phase. The rates of antibody positives were only 2.5% in the population younger than 20 years of age, then rather suddenly shifted up to 70% by 40 years of age, and thereafter maintained a plateau level. These results may indicate relatively quick diminution of hepatitis A, once common in Japan, about 20 years ago and since then a low prevalence has continued until the present time. The change may have been induced by social changes or by the exhaustion of susceptibles after extensive infections with the virus. If the change was induced by such social changes, the present low prevalence is probably stable and will persist for a long time. However, if the change was induced by the exhaustion of the susceptibles, which means that the present is just an interepidemic phase, a large hepatitis A epidemic may recur in future. Another possibility, perhaps the most likely one, is that the change may have been induced by exhaustion of the susceptibles, but later followed by such social changes and such conditions that are unfavorable for spreading hepatitis A virus. If so, the risk of isolated outbreaks due to common infectious sources or small outbreaks due to intimate contacts may increase, but a large epidemic of hepatitis A is less likely to recur as long as such conditions persist. Hepatitis A virus infection was found to be an endemic in Japan but has not been so common during the last 20 years. However, it seems to be still highly prevalent in some developing countries. Therefore, for the people of Japan, especially for young Japanese, the risk of acquiring hepatitis A while visiting such countries has obviously increased in recent years. As a measure to prevent hepatitis A, human immunoglobulin preparations have successfully been used for many years. Woodson and Clinton (1969) reported that Peace Corps volunteers were well protected from hepatitis during their service in developing countries by administration of such a preparation. Recently, Iwarson and Stenqvist (1976) also stated that the Swedish vacationers in the Mediterranean areas were well protected from non-b hepatitis by the administration of human immunoglobulin. Although it is unknown from what type of hepatitis they have been protected, it is a practical measure to protect people from such hepatitis by administration of human immunoglobulin when visiting such developing countries. Twenty-four commercial lots of human immunoglobulin were examined for anti-ha concentration by Miller et al. (1975) employing the IAHA technique. In most lots, titers of 4,000 to 8,000 were detected. Similar experiments were performed in the present study with 16 commercial lots of human immunoglobulin produced in Japan and in most lots, titers of 4,096 were detected. The sensitivity of IAHA varies considerably depending upon the lot of C' or upon the donor of human type-o erythrocytes employed. Using a chimpanzee serum as the measure, we compared the sensitivity of IAHA performed in the present study with that performed in Dr. Purcell's laboratory, and found that

12 336 MORITSUGU et al. Vol. 31 the two IAHA tests were of comparable sensitivities in detection of anti-ha. As stated by Purcell et al. (1976), the sensitivity of their IAHA was equal to that performed in Dr. Hilleman's laboratory. commercial lots of human immunoglobulin Therefore, it was concluded that of both countries contained anti-ha of comparable titers. Since anti-ha positives were rare among young adults, as proposed by Szmuness et al. (1976), it may be better to use plasma materials for immunoglobulin production obtained from donors older than 40 years of age. A viral hepatitis agent other than the hepatitis A or hepatitis B agent was first proposed by Prince et al. (1974), who found that some post-transfusion hepatitis cases showing no evidence of infection with hepatitis B virus were also inconsistent with hepatitis A, i.e., these cases had longer incubation periods and clinical and epidemiological features distinct from those of hepatitis A. Feinstone et al. (1975) stated that non-b transfusion-associated hepatitis cases were serologically unrelated with hepatitis A virus, cytomegalovirus or Epstein- Barr virus. Villarejos et al. (1975) also described non-a, non-b hepatitis occurring in some sporadic and epidemic hepatitis cases found in Costa Rica. All these observations have strongly indicated the existence of an unknown viral agent (or agents) which is currently referred to as non-a non-b hepatitis virus(es). In the present study, many sporadic non-b hepatitis cases were found not due to infection with hepatitis A virus. Although further elucidation is necessary before drawing a final conclusion, it is highly possible that these non-b hepatitis cases determined not due to infection with hepatitis A virus in the present study were caused by such non-a, non-b hepatitis virus(es). If so, not only non-b hepatitis cases associated with blood transfusion but also many sporadic non-b hepatitis cases currently occurring in Japan may be due to infection with such non-a non-b agent(s). ACKNOWLEDGEMENTS We are indebted to Dr. R. H. Purcell, National Institute of Allergy and Infectious Diseases, National Institutes of Health, for hepatitis A antigen and chimpanzee serum; to Dr. F. Ichida,. Niigata University, Dr. A. Tateda, National Sendai Hospital, Dr. H. Suzuki, Dr. K. Mitamura and Dr. T. Oda, Tokyo University Hospital, Dr. T. Katayama, National Tokyo Sanatorium Hospital, Dr. M. Oda, Shinshu University, Dr. Y. Takahashi, Gifu University, Dr. M. Yano, National Nagasaki Hospital, and Dr. R. Kono and Dr. K. Miyamura, WHO Serum Reference Bank, National Institute of Health, for providing serum samples and information; to Mr. K. Takahashi, Jichi Medical School for his technical assistance; and to Dr. I. Tagaya, National. Institute of Health for his interest and support of this study. This investigation was supported in part by grants from the Ministry of Health and Welfare. REFERENCES DIENSTAG, J. L., FEINSTONE, S. M., KAPIKIAN, A. Z., PURCELL, R. H., BOGGS, J. D. AND CONRAD, M. E. (1975a): Fecal shedding of hepatitis-a antigen. Lancet, 1, DIENSTAG, J. L., FEINSTONE, S. M., PURCELL, R. H., HOOFNAGLE, J. H., BARKER, L. F., LONDON, W. T., POPPER, H., PETERSON, J. M. AND KAPIKIAN, A. L. (1975b): Experimental infection of

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