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.
An extensive outbreak of acute hemorrhagic conjunctivitis (AHC) occurred from September to December 1974 in Thailand. At least 29 patients with polio-like motor paralysis that complicated AHC were hospitalized in Bangkok. Paired or triplicate samples of serum from 16 patients were tested for neutralizing antibody to enterovirus type 70 (EV70). A significant rise in titer of antibody was found for two patients, and the other 14 had neutralizing antibody titers ranging from 1:8 to 1:512 without an increasf larger than or equal to 1:16, a level which is considered to be diagnostically significant. Neutralizing antibody to EV70 was detected in 19S fractions of nine sera examined, but neutralizing antibody to three types of poliovirus was confined to 7S fractions. EV70 was isolated from one of seven stool specimens collected on day 37 after the onset of AHC and none of 10 samples of cerebrospinal fluid. These results and additional clinical and epidemiologic findings gave further support to the hypothesis that EV70 infection can cause polio-like motor paralysis as a complication of AHC.
Effect of temperature on growth in monkey kidney (MK) cells of acute hemorrhagic conjunctivitis (AHC) virus was studied. The optimal growth temperature ranged from 32 to 34°, and 39° was nonpermissive. In one-step growth experiments at 33°, the standard strain J 670/71 of AHC virus grew exponentially after a latent period of 2 h, and reached a maximum titer within 6 h post-infection (p.i.). The temperature-shift experiment showed that incubation at 39° during the latent period of the growth cycle had little effect on the subsequent viral growth at 33°, and when the shift-down was carried out at 2 h p. i. or later, viral growth immediately started and reached a maximum. Conversely, viral growth at 33° was immediately stopped by shift-up to the nonpermissive temperature. The shift-down experiment using guanidine-HCl suggested a lack of progeny RNA synthesis at the nonpermissive temperature.
Acute hemorrhagic conjunctivitis (AHC) virus strains isolated in eight different areas during epidemics of AHC were tested for the reproductive capacity at 33, 37 and 39°. All of the 25 strains tested grew better at 33° but restrictively at 39°. The degree of temperature sensitivity varied slightly from one strain to the other, but generally exceeded that of attenuated poliovirus type 1, strain LSc2ab. Temperature-resistant clones were selected by repeated passages of originally temperature-sensitive prototype strains at supra-optimal temperature. The importance of using a low temperature (32–34°) for isolation of virus from external tissues of the body and for subsequent passages has been emphasized. It was suggested that the relatively low temperature of the conjunctiva has played a role in perpetuating temperature sensitivity of this virus.
Human sera were collected in Senegal, Nigeria, Ivory Coast, Dahomey, Liberia, Gabon and Togo during the pre-epidemic period of acute hemorrhagic conjunctivitis (AHC) from 1965 to 1969, and tested for virus neutralizing (VN) antibody to enterovirus type 70 (EV70). Of these, 1109 (91%) were antibody negative (less than equal to 1:4), 116 (9%) neutralized at a dilution of 1:8 or over, and 45 (4%) at dilutions of at least 1:16. The distribution pattern is not significantly different from that of sera collected from Kenya in 1967 or from army recruits in the United States, Argentina, Brazil and Colombia in the 1960s. Sera collected during the post-epidemic period (1970 to 1977) in Senegal, Sierra Leone, Mali, Upper Volta, Chad, Niger and Gabon were also examined; 1573 (68%) were VN antibody negative (less than or equal to 1:4), while 733 (32%) and 433 (19%) had titers of 1:8 or greater and 1:16 or over, respectively. There is a significant difference in distribution between pre- and post-epidemic antibody titers (p less than 0.001), although the incidence of AHC was lower in these countries than in Ghana and Southeast Asia. The prevalence of VN antibodies tends to be lower in the dry, hot inland areas and thus humid coastal monsoonal climates and dense populations seem to favor the spread of AHC.
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