Four serotypes of human rotaviruses that can be differentiated by neutralization tests have been described so far. We prepared serotype 1-, 2-, 3-, and 4-specific, neutralizing monoclonal antibodies to human rotaviruses. All were directed to VP7, a glycoprotein that carries a major serotype specificity. An enzyme-linked immunosorbent assay using these monoclonal antibodies has been developed for serotyping human rotavirus isolates. The sensitivity and specificity of this method were verified by using various strains of human rotavirus that were adapted to cell culture. Furthermore, it was shown that the method was applicable to serotyping human rotaviruses directly in stool specimens.
By employing three strains of cultivable human rotaviruses with different serotype specificity as immunizing antigens, we prepared 11 hybridomas which secreted neutralizing monoclonal antibodies against human rotaviruses. In neutralization tests with four strains of serotype 1, and three each of serotypes 2 and 3, the monoclonal antibodies showed different reactivity patterns: seven monoclonal antibodies reacted specifically with all strains of either serotype 1, 2 or 3 human rotavirus, but two showed strain-specific reactions; the remaining two were commonly reactive to various human rotavirus strains from each serotype but not to two non-human rotaviruses. By immunoprecipitation analysis, it was found that four serotype 2-specific and two commonly reactive antibodies were directed to VP3 (82000 mol. wt. protein) on the outer shell of the virus particles.
Starting with a small amount of diarrheal feces contammg human rotavirus (HRV), we succeeded in propagation of the virus using the roller culture technique with MA-I04 cells. Furthermore, we made a successful adaptation of HRV to a stationary culture and developed a plaque assay for the cell cultureadapted viruses. The 3 culture-adapted virus isolates, KU, YO, and 44 produced plaques (about 0.5-1.0 mm in diameter) under the overlay medium consisting of 0.6% purified agar, 3 fig of acetyl trypsin/ml and 50 pg of DEAE-dextran/ml, Subsequent plaque purification resulted in the formation of clear, larger plaques.It was shown from the results of cross neutralization tests using the fluorescent focus reduction method that the three culture-adapted HRV isolates were clearly different antigenically from bovine rotavirus (NCDV) and, further, that a noticeable difference in antigenicity also existed among the HRV isolates.
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