AMERICAN JOURNAL OF EPIDEMIOLOGY
Vol. 101, No. 5
Copyright © 1975 by The Johns Hopkins University
Printed in U.S.A.
REISAKU KONO, AKIRA SASAGAWA, KIKUKO MIYAMURA AND ETSUKO TAJIRI Kono, R., A. Sasagawa, K. Miyamura. and E. Tajiri (National Institute of Health. Musashimurayama-shi, Tokyo 190-12, Japan). Serologic characterization and sero-epidemiotogic studies on acute hemorrhagic conjunctivitis (AHC) virus. Am J Epidemiol 101:444-457,1975.—Serologic and sero-epidemiologic characteristics of AHC virus infection were studied by neutralization test (NT). Four-fold or greater virus neutralizing (VN) antibody response was demonstrated to the Japanese isolate of AHC virus (the J 670/71 strain) in 77.3% and 66.7% of paired sera from clinical AHC patients in Japan (1971-1973) and Tunisia (1973). The four patients from Indonesia studied in 1972 showed similar antibody response. Cross-neutralization tests of AHC virus isolated in Japan (1971), Taiwan (1971). Hong Kong (1971). Thailand (1972). Indonesia (1972). Singapore (1972). Morocco (1971) and England (1971) with three kinds of antisera prepared against Japanese, Hong Kong and Moroccan AHC virus isolates indicated their antigenic identity. However, isolates from Singapore in 1970 (Singapore 70 virus) were not neutralized with the AHC virus antisera mentioned above: Singapore 70 virus constitutes another antigenic type, to which, however, no VN antibody rise was found in paired patients' sera from Japan, Tunisia and Indonesia. Thus, no serologic evidence supporting an etiologic role of this virus group in the development of AHC was found. Although cross-tests using monospecific antisera suggested some cross-relation between AHC and both echovirus type 4 (E4) and coxsackie A (CA). type 19, no serologic relationship between AHC and these viruses was found. Sera from healthy individuals collected before and after AHC outbreaks were tested for VN antibody against AHC virus in Japan and two epidemic foci. Ghana and Indonesia. Before the epidemic. 80 to 90% of the people lacked antibody in the three countries, but 39.7% and 45.2% of inhabitants posessed VN antibody of 1:8 or over in Ghana and Indonesia after the outbreak. In Japan, however, only a slight increase was found in VN antibody prevalence afterwards. Serologic study showed that 41.5% of horse sera were VN positive at dilutions of 1:8 or more; many cattle sera also had a low VN titer but few cynomologus monkey sera had VN activity. Conjunctivitis; eye diseases INTRODUCTION subcontinent, Asia and some European A pandemic of acute hemorrhagic con- countries from 1969 to 1974. It has now junctivitis (AHC) swept Africa, the Indian Supported by a grant in aid from the Ministry of Received for publication October 21, 1974, and in final form December 16, 1974. Abbreviations: AHC, acute hemorrhagic conjunctivitis; CA, coxsackie A virus; CA1-24, coxsackie A virus types 1-24; E4, echovirus type 4; MK, monkey kidney; NT, neutralization test; VN, virus neutralizing. 'Central Virus Diagnostic Laboratory, National Institute of Health, Tokyo 3260 Nakato Musashimurayama-shi Tokyo 190-12, Japan. (Address for reprint requests which should be sent to R. Kono.)
Education. We are indebted to Prof. S. Hotta and Indonesian scientists (see reference 13) for sera collected in Surabaya 1968, and to Prof. M. Kanamitsu, Dr. T. Ogata and Dr. J. Sulianti Saroso and other Indonesian doctors who shared and permitted us to use part of sera collected in Pontianak 1972 for the present study. We are grateful to Prof. K. Minami, Fukushima Medical School; Dr. C. 0. Quarcoopome, Dr. S. N. Afoaka and Dr. P. K. A. Addy; University of Ghana Medical School; Prof. R. Mabrouk and Prof, T. Daghfous, l'lnstitut d'Ophthalmologie, Tunis, for their serum collections.
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SEROLOGIC CHARACTERIZATION AND SERO-EPIDEMIOLOGIC STUDIES ON ACUTE HEMORRHAGIC CONJUNCTIVITIS (AHC) VIRUS1
ACUTE HEMORRHAGIC CONJUNCTIVITIS VIRUS
become a common eye infection in these areas which breaks out from time to time in different locales. In 1971 we successfully isolated the causative virus from the conjunctival scrapings or swabs of Japanese patients with AHC and reported that it has the general characteristics of an enterovirus, but that intersecting pools of antisera against known types of enterovirus failed to neutralize it (1). This finding has been confirmed by several other investigators (2-6) and AHC virus is classified by collaborative studies as enterovirus type 70 (6). In this paper, we describe the results of supplementary serologic characterization of AHC virus and sero-epidemiologic studies of its infection in Japan, Indonesia and Ghana.
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strains were isolated in HeLa cell culture except the English ones which were isolated in human fetal trachea organ culture and were adapted to primary MK cell culture in this laboratory for further studies. Singapore 70 virus. The TW 18 and EH 36 strains, which had been isolated in the epidemic of conjunctivitis in 1970, were obtained from Dr. M. Yin-Murphy (8) and were adapted to MK and then used in this study. These strains are grouped as Singapore 70 virus in this report because their antigenic structure was proved to be different from other AHC virus. Some other isolates from Singapore, 1970, and one strain from Hong Kong (the HK 3751 strain), 1971, could not be adapted to MK cell culture and therefore were excluded MATERIALS AND METHODS from the study. According to Yin-Murphy, all these strains were reported to be like Viruses TW 18 and EH 36 and belonged to the AHC virus. Two Japanese isolates in Singapore 70 virus group (8). 1971, the J 648/71 and J 670/71 strains, Other viruses. The Pesascek and Du Toit were used; the latter was more often used strains of echovirus type 4 (E4) were obas the standard strain. The two strains tained from Dr. M. Nakano, Department were isolated from conjunctival scrapings of Enteroviruses of this Institute and were of AHC patients in human embryonic propagated in MK or human embryonic kidney cell culture, they were adapted to lung (HEL) cell culture in this laboratory. cynomologus monkey kidney (MK) cell Coxsackie A virus type 19 (CA19), the culture, and their antigen identity was Dohi strain, was obtained from Dr. I. established (1). Tagaya, Director, Department of EnThe other AHC virus isolates used were: terovirus, National Institute of Health, 1) Moroccan isolates in 1971, the Rabat 6 Tokyo, and was propagated in subcutaneand 20 strains from Prof. R. Sohier (3); 2) ously inoculated one-day-old suckling Hong Kong isolates in 1971, the HK 3454 mice. strain from Dr. W. K. Chang, Queen Mary Hospital, Hong Kong; 3) Taiwan isolates in 1971, the T 435 and T 471 strains from Drs. Sera from patients with clinical AHC and E4 virus infections M. H. Hatch and J. C. Hierholzer, Center for Disease Control (CDC), Atlanta, Thai Seventy-five pairs of acute and convalesisolate in 1972, the AE 7 strain from Dr. N. cent sera were collected from clinical AHC Sangkawibha (7); 5) Indonesian isolate in cases during the period from December 1972, the Indo/13476/72 strain from Dr. I. 1971 to September 1973 in Tokyo, HokJ. Green, NAMRU 2, Taipei; 6) Singapore kaido, Gunma, Mie and Hiroshima prefecisolate in 1972, the SEC 57/72 strain from tures, Japan: 24 pairs in 1971; 27 pairs in Dr. M. Yin-Murphy, University of Sin- 1972; and 24 pairs in 1973. gapore, and 7) English isolates in 1971, the Four pairs of sera taken in Djakarta, Eng/23356/71 and Eng/20254/71 strains 1972, were sent for testing by Dr. M. from Prof. B. R. Jones (4, 5). All these Marsetio from Djakarta, Indonesia.
446
KONO, SASAGAWA, MIYAMURA AND TAJIRI
Laboratory, (CVDL), Tokyo. Antisera against the Du Toit, Shirosphire, KP, and 197 strains were prepared in guinea pigs in the CVDL (the latter two strains are Japanese E4 isolates) (8). CA antisera. CA horse antisera prepared at NIH, Bethesda, were used (10). Typespecific antisera for CA type 1 through 24 except types 3 and 17 were available. Also, Antisera to the isolates from patients with preimmunization sera from horses immuconjunctivitis in Japan and elsewhere nized with CA types 2, 8, 10, 11, 13-16, Monkey antiserum to the J 670/71 strain 18-21, and 24 were used as controls in the (our representative Japanese AHC virus). neutralization tests. We described previously (9) how to prepare CA19 mouse antiserum (No. 19981) and inoculum of the J 670/71 strain for the hamster antiserum (S-2179) were supplied neurovirulence test in monkeys. Since one through the courtesy of Dr. L. Rosen and of the monkeys whose VN antibody titer Dr. N. Schmidt, respectively. was 1:32 on day 14 post infection survived with left hind leg paralysis in the neurovirSera from healthy individuals ulence test (9), it was inoculated once more Two sets of serum collections before and by intravenous injection of 106-6 TCID50 of after the AHC epidemic were obtained in the same purified virus inoculum on day Japan, Ghana and Indonesia. 14, and bled on day 21 post second inoculaJapanese sera. Two hundred forty-four tion. The VN antibody titer against about sera from healthy Japanese in Gunma 100 TCID50 of homologous strain of AHC prefecture and vicinity (Chiba and Saivirus was 1:5,120 to 1:10,240. tama prefectures) were randomly selected Rabat 11 (Moroccan AHC virus) and HK in 1970 by computer from our Serum Refer3454 (Hong Kong AHC virus). Monkey ence Bank collection before the AHC epiantisera to these other AHC virus strains demic. Another 243 sera were grab samples were generously supplied to us by Prof. R. collected in Gunma in 1972 after the introSohier and Dr. W. K. Chang. Singapore 70 virus, the EH 24 strain (a duction of AHC virus in Japan. Ghanaian sera. Ghanaian sera were colviral isolate in Singapore, 1970) and the lected originally by Prof. Minami and GhaHK 3751 strain. Monkey antisera to these naian scientists for study of Australia antiviruses were kindly given to us by Dr. M. gen and other purposes (12). Therefore, Yin-Murphy and Dr. W. K. Chang. they were not "normal" sera in the strict Enterovirus antisera sense, but it may be permissible to regard E4 antisera. Five antisera against the them as "normal" in relation to AHC Pesascek strain were tested: 1) Lot 3 of because the donors did not have conjunctirabbit antiserum from CDC, 2) type 4 vitis at the time they were bled. (Pesascek) ECHO antiserum (monkey) Thirty-nine sera were collected before prepared by the National Foundation In- the epidemic at Korle Bu Hospital Accra, fantile Paralysis, 3) antiserum prepared at in December 1968. Thirty-one sera were NIH, Tokyo, 4) horse antiserum prepared from the Upper North Region where no at NIH, Bethesda, under contract with epidemic was recognized. Sixty-three sera WHO (10), and 5) guinea pig antiserum were collected during the period from May prepared in the Central Virus Diagnostic 1970 to June 1971 in Accra and its neigh-
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Twenty-one pairs were collected by Dr. R. Mabrouk in Tunis, 1973, and also were sent to us for serologic tests. Five pairs of sera from patients with E4 virus infection which had been stored in this laboratory were tested for VN antibody to AHC virus. All the serum pairs were stored at -70 C until use.
ACUTE HEMORRHAGIC CONJUNCTIVITIS VIRUS
447
Neutralization test in cell culture The neutralization test (NT) with AHC virus was performed in microplate cultures of primary MK cells. Serial two-fold dilution of each serum was made with diluter in a volume of 25 n\ in wells of "piggyback"-type transfer plate and the same volume of virus fluid containing 100 TCID50 was added to each well (14). After incubation periods of 1 hour at 37 C and overnight at 4 C, the transfer plates were placed on a previously prepared microplate with complete MK cell sheets on the bottom of each well and 100 ^1 of maintenance medium. The microplates were incubated at 33 C for AHC virus, and read for any resulting cytopathic effect using an inverted microscope 4 to 5 days postinfection. The growth medium was Eagle's minimum essential medium (MEM) plus 10 per cent bovine se-
RESULTS
Serologic response of patients with clinical AHC Neutralization tests. NT.with AHC virus was performed on 75 pairs of acute and convalescent sera from patients from different parts of Japan who were clinically diagnosed as having AHC from December 1971 to September 1973. Fifty-eight (77.3 per cent) revealed four-fold or greater rise of VN antibody titers against AHC virus: 19 of 24 pairs in 1971, 22 of 27 pairs in 1972; and 17 of 24 pairs in 1973 showed four-fold or greater antibody rise to AHC virus. Figure 1 depicts the pattern of antibody rise in those patients. The results of NT of four pairs of Indonesian and 21 pairs of Tunisian patients' sera collected in 1972 and 1973, respectively, are included also in figure 1. All four of the
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boring epidemic areas at a time when AHC rum and the maintenance medium was outbreak was definitely over. Eagle's MEM plus 0.1 per cent bovine Indonesian sera. One hundred eleven albumin fraction V. pre-epidemic sera were collected by Prof. In the comparison of AHC virus with E4 S. Hotta and Indonesian doctors in Sura- virus, we used a HEL cell microplate baya, East Java, 1968 (13). Three hundred culture for NT with E4 virus, since, accordfifty-six post-epidemic sera were collected ing to our own and other (11) experience, by Prof. M. Kanamitsu and Dr. T. Ogata NT end point could be clearly shown in from inhabitants of Pontianak, Kaliman- HEL cell culture with either the Pesascek tan (West Borneo), in 1972. The above two or Du Toit strain of E4 virus. Except for collections were made for arbovirus sero- higher incubation temperature (37 C), the epidemiology. Although coming from dif- NT procedure was the same as above. ferent, widely separated populations, they Neutralization test in suckling mice provide the best available sources for data Suckling mouse neutralization tests of concerning AHC antibody prevalence beCA19 (the Dohi strain) with J 670/71 antifore and after the AHC epidemic which serum and CA19 horse antiserum swept through all of Indonesia in 1971. (V-022-501-560) were performed simultaneSera from normal animals ously. The serum-virus mixtures which One hundred eighteen horse sera, sup- contained equal volumes of two-fold seriplied through the courtesy of Dr. Y. Kono, ally diluted serum and virus (300 LD50) National Institute of Animal Health, To- were incubated at 37 C for 1 hr, after kyo, were collected in Japan; 22 sera from having been kept overnight at 4 C and then normal cattle were obtained from Chiba inoculated in 0.04-ml aliquots subcutaneSerum Institute, Japan; and 48 sera of ously into 1-day-old suckling mice. Five normal cynomologus monkeys were ob- animals were used per dilution, and they were kept under observation for 3 weeks. tained from Malaysia and Indonesia.
448
KONO, SASAGAWA, MIYAMURA AND TAJIRI
1021 14.
256
4 a*o
t.
It*
** •
128
m
•n
••a
o'
*:
32
^ /
••
16 /
8
/
1
t
Japanese
O
Tunisian
A
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Titers equal or over
32 61 128
FIGURE 1. Neutralizing antibody titers against AHC virus in individual patients with acute hemorrhagic conjunctivitis comparing acute and convalescent sera in Japan, Indonesia and Tunisia.
Indonesian and 14 of 21 Tunisian pairs (66.7 per cent) also revealed four-fold or greater antibody rise to Japanese AHC virus (the J 670/71 strain). The results clearly confirmed that AHC virus isolated by us in 1971 is a causative agent of AHC which has been occurring since 1971 in these three countries. In the above results nine patients had a VN titer of 1:8 in their second serum; of these three had a four-fold rise, three showed a two-fold rise, and the other three showed no rise. Accordingly, most patients responded to AHC with VN antibody titers of 1:16 or over. Therefore, a VN antibody titer of 1:16 is considered as the critical diagnostic level. In the following seroepidemiologic studies, however, we accepted a titer of 1:8 as the lowest specific level of VN antibody indicating past exposure to AHC virus because we wanted to detect the maximum number of responders. The geometric mean VN antibody titers were calculated as 1:83.8 in
Cross-neutralization test of different isolates The results of cross-neutralization test of the isolates from AHC cases in Japan (1971), Singapore (1970 and 1972), Hong Kong (1971), Taiwan (1971), Thailand (1972), Indonesia (1972), Morocco (1971) and England (1971) with antisera against viral strains from Japan (the J 670/71 strain), Singapore (the EH 24 strain), Hong Kong (the HK 3454 and HK 3751 strains) and Morocco (the Rabat 11 strain) are shown in table 2. It was clearly demonstrated that the Singapore isolates in 1970 are antigenically different from the others and one of the Hong Kong isolates (the HK 3751 strain) probably belongs to the same antigenic group; on the other hand, all the other isolates in Asia, Africa and Europe are closely related. Together with the results of NT using patients' paired sera mentioned above, it was concluded that the pandemic virus belongs to one antigenic type represented by the first isolate in Japan, the J 670/71 strain.
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512
patients who showed four-fold or more antibody rise in their convalescence. Neutralization test of Singapore 70 virus. Table 1 compares the results of NT of Singapore 70 virus (the EH 36 strain) and of AHC virus with paired sera from different parts of Japan, Djakarta and Tunis. We selected 15 paired sera serologic responders to AHC virus from Tokyo, Mie and Hokkaido, Japan; and from Djakarta and Tunis. Although all showed distinct antibody responses to AHC virus, none revealed VN antibody rises to EH 36 virus. There were some non-responders among patients with clinical AHC, who, it might be expected, could have been infected with a different virus, possibly Singapore 70 virus. We tested five such pairs from Tunisian sera but none of them showed serologic evidence of EH 36 virus infection.
449
ACUTE HEM0RRHAG1C CONJUNCTIVITIS VIRUS TABLE 1
Results of neutralization test of Singapore 70 virus (EH 36 strain) with paired AHC patient's sera from different parts of the world
Groups
Serologic responders Djakarta Tokyo, Japan
Mie, Japan Hokkaido, Japan Tunis
No.
1
2 3 1 2 3 4 5 6 7 1 2
3 Serologic non-responders Tunis
4 5 6 7 8 9 10
Acute
8*
Vol. 101, No. 5
Copyright © 1975 by The Johns Hopkins University
Printed in U.S.A.
REISAKU KONO, AKIRA SASAGAWA, KIKUKO MIYAMURA AND ETSUKO TAJIRI Kono, R., A. Sasagawa, K. Miyamura. and E. Tajiri (National Institute of Health. Musashimurayama-shi, Tokyo 190-12, Japan). Serologic characterization and sero-epidemiotogic studies on acute hemorrhagic conjunctivitis (AHC) virus. Am J Epidemiol 101:444-457,1975.—Serologic and sero-epidemiologic characteristics of AHC virus infection were studied by neutralization test (NT). Four-fold or greater virus neutralizing (VN) antibody response was demonstrated to the Japanese isolate of AHC virus (the J 670/71 strain) in 77.3% and 66.7% of paired sera from clinical AHC patients in Japan (1971-1973) and Tunisia (1973). The four patients from Indonesia studied in 1972 showed similar antibody response. Cross-neutralization tests of AHC virus isolated in Japan (1971), Taiwan (1971). Hong Kong (1971). Thailand (1972). Indonesia (1972). Singapore (1972). Morocco (1971) and England (1971) with three kinds of antisera prepared against Japanese, Hong Kong and Moroccan AHC virus isolates indicated their antigenic identity. However, isolates from Singapore in 1970 (Singapore 70 virus) were not neutralized with the AHC virus antisera mentioned above: Singapore 70 virus constitutes another antigenic type, to which, however, no VN antibody rise was found in paired patients' sera from Japan, Tunisia and Indonesia. Thus, no serologic evidence supporting an etiologic role of this virus group in the development of AHC was found. Although cross-tests using monospecific antisera suggested some cross-relation between AHC and both echovirus type 4 (E4) and coxsackie A (CA). type 19, no serologic relationship between AHC and these viruses was found. Sera from healthy individuals collected before and after AHC outbreaks were tested for VN antibody against AHC virus in Japan and two epidemic foci. Ghana and Indonesia. Before the epidemic. 80 to 90% of the people lacked antibody in the three countries, but 39.7% and 45.2% of inhabitants posessed VN antibody of 1:8 or over in Ghana and Indonesia after the outbreak. In Japan, however, only a slight increase was found in VN antibody prevalence afterwards. Serologic study showed that 41.5% of horse sera were VN positive at dilutions of 1:8 or more; many cattle sera also had a low VN titer but few cynomologus monkey sera had VN activity. Conjunctivitis; eye diseases INTRODUCTION subcontinent, Asia and some European A pandemic of acute hemorrhagic con- countries from 1969 to 1974. It has now junctivitis (AHC) swept Africa, the Indian Supported by a grant in aid from the Ministry of Received for publication October 21, 1974, and in final form December 16, 1974. Abbreviations: AHC, acute hemorrhagic conjunctivitis; CA, coxsackie A virus; CA1-24, coxsackie A virus types 1-24; E4, echovirus type 4; MK, monkey kidney; NT, neutralization test; VN, virus neutralizing. 'Central Virus Diagnostic Laboratory, National Institute of Health, Tokyo 3260 Nakato Musashimurayama-shi Tokyo 190-12, Japan. (Address for reprint requests which should be sent to R. Kono.)
Education. We are indebted to Prof. S. Hotta and Indonesian scientists (see reference 13) for sera collected in Surabaya 1968, and to Prof. M. Kanamitsu, Dr. T. Ogata and Dr. J. Sulianti Saroso and other Indonesian doctors who shared and permitted us to use part of sera collected in Pontianak 1972 for the present study. We are grateful to Prof. K. Minami, Fukushima Medical School; Dr. C. 0. Quarcoopome, Dr. S. N. Afoaka and Dr. P. K. A. Addy; University of Ghana Medical School; Prof. R. Mabrouk and Prof, T. Daghfous, l'lnstitut d'Ophthalmologie, Tunis, for their serum collections.
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SEROLOGIC CHARACTERIZATION AND SERO-EPIDEMIOLOGIC STUDIES ON ACUTE HEMORRHAGIC CONJUNCTIVITIS (AHC) VIRUS1
ACUTE HEMORRHAGIC CONJUNCTIVITIS VIRUS
become a common eye infection in these areas which breaks out from time to time in different locales. In 1971 we successfully isolated the causative virus from the conjunctival scrapings or swabs of Japanese patients with AHC and reported that it has the general characteristics of an enterovirus, but that intersecting pools of antisera against known types of enterovirus failed to neutralize it (1). This finding has been confirmed by several other investigators (2-6) and AHC virus is classified by collaborative studies as enterovirus type 70 (6). In this paper, we describe the results of supplementary serologic characterization of AHC virus and sero-epidemiologic studies of its infection in Japan, Indonesia and Ghana.
445
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strains were isolated in HeLa cell culture except the English ones which were isolated in human fetal trachea organ culture and were adapted to primary MK cell culture in this laboratory for further studies. Singapore 70 virus. The TW 18 and EH 36 strains, which had been isolated in the epidemic of conjunctivitis in 1970, were obtained from Dr. M. Yin-Murphy (8) and were adapted to MK and then used in this study. These strains are grouped as Singapore 70 virus in this report because their antigenic structure was proved to be different from other AHC virus. Some other isolates from Singapore, 1970, and one strain from Hong Kong (the HK 3751 strain), 1971, could not be adapted to MK cell culture and therefore were excluded MATERIALS AND METHODS from the study. According to Yin-Murphy, all these strains were reported to be like Viruses TW 18 and EH 36 and belonged to the AHC virus. Two Japanese isolates in Singapore 70 virus group (8). 1971, the J 648/71 and J 670/71 strains, Other viruses. The Pesascek and Du Toit were used; the latter was more often used strains of echovirus type 4 (E4) were obas the standard strain. The two strains tained from Dr. M. Nakano, Department were isolated from conjunctival scrapings of Enteroviruses of this Institute and were of AHC patients in human embryonic propagated in MK or human embryonic kidney cell culture, they were adapted to lung (HEL) cell culture in this laboratory. cynomologus monkey kidney (MK) cell Coxsackie A virus type 19 (CA19), the culture, and their antigen identity was Dohi strain, was obtained from Dr. I. established (1). Tagaya, Director, Department of EnThe other AHC virus isolates used were: terovirus, National Institute of Health, 1) Moroccan isolates in 1971, the Rabat 6 Tokyo, and was propagated in subcutaneand 20 strains from Prof. R. Sohier (3); 2) ously inoculated one-day-old suckling Hong Kong isolates in 1971, the HK 3454 mice. strain from Dr. W. K. Chang, Queen Mary Hospital, Hong Kong; 3) Taiwan isolates in 1971, the T 435 and T 471 strains from Drs. Sera from patients with clinical AHC and E4 virus infections M. H. Hatch and J. C. Hierholzer, Center for Disease Control (CDC), Atlanta, Thai Seventy-five pairs of acute and convalesisolate in 1972, the AE 7 strain from Dr. N. cent sera were collected from clinical AHC Sangkawibha (7); 5) Indonesian isolate in cases during the period from December 1972, the Indo/13476/72 strain from Dr. I. 1971 to September 1973 in Tokyo, HokJ. Green, NAMRU 2, Taipei; 6) Singapore kaido, Gunma, Mie and Hiroshima prefecisolate in 1972, the SEC 57/72 strain from tures, Japan: 24 pairs in 1971; 27 pairs in Dr. M. Yin-Murphy, University of Sin- 1972; and 24 pairs in 1973. gapore, and 7) English isolates in 1971, the Four pairs of sera taken in Djakarta, Eng/23356/71 and Eng/20254/71 strains 1972, were sent for testing by Dr. M. from Prof. B. R. Jones (4, 5). All these Marsetio from Djakarta, Indonesia.
446
KONO, SASAGAWA, MIYAMURA AND TAJIRI
Laboratory, (CVDL), Tokyo. Antisera against the Du Toit, Shirosphire, KP, and 197 strains were prepared in guinea pigs in the CVDL (the latter two strains are Japanese E4 isolates) (8). CA antisera. CA horse antisera prepared at NIH, Bethesda, were used (10). Typespecific antisera for CA type 1 through 24 except types 3 and 17 were available. Also, Antisera to the isolates from patients with preimmunization sera from horses immuconjunctivitis in Japan and elsewhere nized with CA types 2, 8, 10, 11, 13-16, Monkey antiserum to the J 670/71 strain 18-21, and 24 were used as controls in the (our representative Japanese AHC virus). neutralization tests. We described previously (9) how to prepare CA19 mouse antiserum (No. 19981) and inoculum of the J 670/71 strain for the hamster antiserum (S-2179) were supplied neurovirulence test in monkeys. Since one through the courtesy of Dr. L. Rosen and of the monkeys whose VN antibody titer Dr. N. Schmidt, respectively. was 1:32 on day 14 post infection survived with left hind leg paralysis in the neurovirSera from healthy individuals ulence test (9), it was inoculated once more Two sets of serum collections before and by intravenous injection of 106-6 TCID50 of after the AHC epidemic were obtained in the same purified virus inoculum on day Japan, Ghana and Indonesia. 14, and bled on day 21 post second inoculaJapanese sera. Two hundred forty-four tion. The VN antibody titer against about sera from healthy Japanese in Gunma 100 TCID50 of homologous strain of AHC prefecture and vicinity (Chiba and Saivirus was 1:5,120 to 1:10,240. tama prefectures) were randomly selected Rabat 11 (Moroccan AHC virus) and HK in 1970 by computer from our Serum Refer3454 (Hong Kong AHC virus). Monkey ence Bank collection before the AHC epiantisera to these other AHC virus strains demic. Another 243 sera were grab samples were generously supplied to us by Prof. R. collected in Gunma in 1972 after the introSohier and Dr. W. K. Chang. Singapore 70 virus, the EH 24 strain (a duction of AHC virus in Japan. Ghanaian sera. Ghanaian sera were colviral isolate in Singapore, 1970) and the lected originally by Prof. Minami and GhaHK 3751 strain. Monkey antisera to these naian scientists for study of Australia antiviruses were kindly given to us by Dr. M. gen and other purposes (12). Therefore, Yin-Murphy and Dr. W. K. Chang. they were not "normal" sera in the strict Enterovirus antisera sense, but it may be permissible to regard E4 antisera. Five antisera against the them as "normal" in relation to AHC Pesascek strain were tested: 1) Lot 3 of because the donors did not have conjunctirabbit antiserum from CDC, 2) type 4 vitis at the time they were bled. (Pesascek) ECHO antiserum (monkey) Thirty-nine sera were collected before prepared by the National Foundation In- the epidemic at Korle Bu Hospital Accra, fantile Paralysis, 3) antiserum prepared at in December 1968. Thirty-one sera were NIH, Tokyo, 4) horse antiserum prepared from the Upper North Region where no at NIH, Bethesda, under contract with epidemic was recognized. Sixty-three sera WHO (10), and 5) guinea pig antiserum were collected during the period from May prepared in the Central Virus Diagnostic 1970 to June 1971 in Accra and its neigh-
Downloaded from https://academic.oup.com/aje/article-abstract/101/5/444/120435 by Western Sydney University Library user on 13 January 2019
Twenty-one pairs were collected by Dr. R. Mabrouk in Tunis, 1973, and also were sent to us for serologic tests. Five pairs of sera from patients with E4 virus infection which had been stored in this laboratory were tested for VN antibody to AHC virus. All the serum pairs were stored at -70 C until use.
ACUTE HEMORRHAGIC CONJUNCTIVITIS VIRUS
447
Neutralization test in cell culture The neutralization test (NT) with AHC virus was performed in microplate cultures of primary MK cells. Serial two-fold dilution of each serum was made with diluter in a volume of 25 n\ in wells of "piggyback"-type transfer plate and the same volume of virus fluid containing 100 TCID50 was added to each well (14). After incubation periods of 1 hour at 37 C and overnight at 4 C, the transfer plates were placed on a previously prepared microplate with complete MK cell sheets on the bottom of each well and 100 ^1 of maintenance medium. The microplates were incubated at 33 C for AHC virus, and read for any resulting cytopathic effect using an inverted microscope 4 to 5 days postinfection. The growth medium was Eagle's minimum essential medium (MEM) plus 10 per cent bovine se-
RESULTS
Serologic response of patients with clinical AHC Neutralization tests. NT.with AHC virus was performed on 75 pairs of acute and convalescent sera from patients from different parts of Japan who were clinically diagnosed as having AHC from December 1971 to September 1973. Fifty-eight (77.3 per cent) revealed four-fold or greater rise of VN antibody titers against AHC virus: 19 of 24 pairs in 1971, 22 of 27 pairs in 1972; and 17 of 24 pairs in 1973 showed four-fold or greater antibody rise to AHC virus. Figure 1 depicts the pattern of antibody rise in those patients. The results of NT of four pairs of Indonesian and 21 pairs of Tunisian patients' sera collected in 1972 and 1973, respectively, are included also in figure 1. All four of the
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boring epidemic areas at a time when AHC rum and the maintenance medium was outbreak was definitely over. Eagle's MEM plus 0.1 per cent bovine Indonesian sera. One hundred eleven albumin fraction V. pre-epidemic sera were collected by Prof. In the comparison of AHC virus with E4 S. Hotta and Indonesian doctors in Sura- virus, we used a HEL cell microplate baya, East Java, 1968 (13). Three hundred culture for NT with E4 virus, since, accordfifty-six post-epidemic sera were collected ing to our own and other (11) experience, by Prof. M. Kanamitsu and Dr. T. Ogata NT end point could be clearly shown in from inhabitants of Pontianak, Kaliman- HEL cell culture with either the Pesascek tan (West Borneo), in 1972. The above two or Du Toit strain of E4 virus. Except for collections were made for arbovirus sero- higher incubation temperature (37 C), the epidemiology. Although coming from dif- NT procedure was the same as above. ferent, widely separated populations, they Neutralization test in suckling mice provide the best available sources for data Suckling mouse neutralization tests of concerning AHC antibody prevalence beCA19 (the Dohi strain) with J 670/71 antifore and after the AHC epidemic which serum and CA19 horse antiserum swept through all of Indonesia in 1971. (V-022-501-560) were performed simultaneSera from normal animals ously. The serum-virus mixtures which One hundred eighteen horse sera, sup- contained equal volumes of two-fold seriplied through the courtesy of Dr. Y. Kono, ally diluted serum and virus (300 LD50) National Institute of Animal Health, To- were incubated at 37 C for 1 hr, after kyo, were collected in Japan; 22 sera from having been kept overnight at 4 C and then normal cattle were obtained from Chiba inoculated in 0.04-ml aliquots subcutaneSerum Institute, Japan; and 48 sera of ously into 1-day-old suckling mice. Five normal cynomologus monkeys were ob- animals were used per dilution, and they were kept under observation for 3 weeks. tained from Malaysia and Indonesia.
448
KONO, SASAGAWA, MIYAMURA AND TAJIRI
1021 14.
256
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128
m
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8
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Japanese
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Tunisian
A
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Titers equal or over
32 61 128
FIGURE 1. Neutralizing antibody titers against AHC virus in individual patients with acute hemorrhagic conjunctivitis comparing acute and convalescent sera in Japan, Indonesia and Tunisia.
Indonesian and 14 of 21 Tunisian pairs (66.7 per cent) also revealed four-fold or greater antibody rise to Japanese AHC virus (the J 670/71 strain). The results clearly confirmed that AHC virus isolated by us in 1971 is a causative agent of AHC which has been occurring since 1971 in these three countries. In the above results nine patients had a VN titer of 1:8 in their second serum; of these three had a four-fold rise, three showed a two-fold rise, and the other three showed no rise. Accordingly, most patients responded to AHC with VN antibody titers of 1:16 or over. Therefore, a VN antibody titer of 1:16 is considered as the critical diagnostic level. In the following seroepidemiologic studies, however, we accepted a titer of 1:8 as the lowest specific level of VN antibody indicating past exposure to AHC virus because we wanted to detect the maximum number of responders. The geometric mean VN antibody titers were calculated as 1:83.8 in
Cross-neutralization test of different isolates The results of cross-neutralization test of the isolates from AHC cases in Japan (1971), Singapore (1970 and 1972), Hong Kong (1971), Taiwan (1971), Thailand (1972), Indonesia (1972), Morocco (1971) and England (1971) with antisera against viral strains from Japan (the J 670/71 strain), Singapore (the EH 24 strain), Hong Kong (the HK 3454 and HK 3751 strains) and Morocco (the Rabat 11 strain) are shown in table 2. It was clearly demonstrated that the Singapore isolates in 1970 are antigenically different from the others and one of the Hong Kong isolates (the HK 3751 strain) probably belongs to the same antigenic group; on the other hand, all the other isolates in Asia, Africa and Europe are closely related. Together with the results of NT using patients' paired sera mentioned above, it was concluded that the pandemic virus belongs to one antigenic type represented by the first isolate in Japan, the J 670/71 strain.
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512
patients who showed four-fold or more antibody rise in their convalescence. Neutralization test of Singapore 70 virus. Table 1 compares the results of NT of Singapore 70 virus (the EH 36 strain) and of AHC virus with paired sera from different parts of Japan, Djakarta and Tunis. We selected 15 paired sera serologic responders to AHC virus from Tokyo, Mie and Hokkaido, Japan; and from Djakarta and Tunis. Although all showed distinct antibody responses to AHC virus, none revealed VN antibody rises to EH 36 virus. There were some non-responders among patients with clinical AHC, who, it might be expected, could have been infected with a different virus, possibly Singapore 70 virus. We tested five such pairs from Tunisian sera but none of them showed serologic evidence of EH 36 virus infection.
449
ACUTE HEM0RRHAG1C CONJUNCTIVITIS VIRUS TABLE 1
Results of neutralization test of Singapore 70 virus (EH 36 strain) with paired AHC patient's sera from different parts of the world
Groups
Serologic responders Djakarta Tokyo, Japan
Mie, Japan Hokkaido, Japan Tunis
No.
1
2 3 1 2 3 4 5 6 7 1 2
3 Serologic non-responders Tunis
4 5 6 7 8 9 10
Acute
8*
