|Budget Amount *help
¥2,100,000 (Direct Cost : ¥2,100,000)
Fiscal Year 1990 : ¥900,000 (Direct Cost : ¥900,000)
Fiscal Year 1989 : ¥1,200,000 (Direct Cost : ¥1,200,000)
1. The ability of influenza C virus to destroy the receptors on chicken erythrocytes and the inhibitory activity of rat serum inhibitors was inhibited efficiently by the monoclonal antibodies to site A-1 (J14, J9, Q5) but not by those site A-2 (K16) and site B-1 (S16), which were derived to HE glycoprotein of C/Ann Arbor/1/50 virus. Of the three antibodies to site A-1, J14 showed the highest inhibitory activity. Thus the RDE site of influenza C virus may be located closest to the epitope recognized by J14.
2. The removal of O-acetyl groups from either 9-0-acetyl-N-acetylneuraminic acid or p-nitrophenyl acetate, caused by the viral RDE, was not prevented at all by any of the monoclonal antibodies tested. Furthermore, none of several polyclonal antiviral sera prepared in different animal species was able to block the hydrolysis of these small substrates, raising the possibility that the catalytic site of influenza C virus RDE is antigenically silent.
3. SDS-PAGE (reducing conditions) analy
sis of ^3H-DFP labeled HE glycoprotein revealed that the DFP binding site (active serin residue of RDE) was localized in HE1 subunit. The labeled glycoprotein was digested with CNBr, and the peptide having appropriate molecular size of 6,000 which is co ntaining the ^3H-DFP was obtained from the digestion products by the gel filtration and reverse phase HPLC. Further purification and amino acid sequencing of the corresponding peptide are now carrying on.
4. All of the 16 mutants of C/Ann Arbor/1/50 virus, which were escaped from neutralizing action of the anti-HE monoclonal antibodies, were found to possess the different HA and RDE activities compared to those of. the parent virus. Nucleotide sequencing analysis of HE genes of these mutants revealed that mutations in the amino acids 245, 266, 283, (266, 283 in particular) of the HE glycoprotein resulted in increasing, and mutations in the amino acids 186, 187, 190, 206, 212, 217, 226 (186, 187 in particular) resulted in decreasing the receptor recognizability of the virus.