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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 Sinapore 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.