Rabies virulence: effect on pathogenicity and sequence characterization of rabies virus mutations affecting antigenic...

Using four neutralizing monoclonal antibodies which presumably bind to the same antigenic site on the CVS glycoprotein (antigenic site III as defined by cross-neutralization tests), we isolated 58 mutants of the CVS strain of rabies virus. These mutants were highly resistant to the selecting antibodies and grew efficiently in cell cultures. We classified them into five groups on the basis of the pattern of resistance to the four antibodies. We determined pathogenicities of the mutants for adult mice by intracerebral inoculation. Group 2 mutants were nonpathogenic or had attenuated pathogenicity. On the contrary, mutants from the other groups were pathogenic, causing paralysis and death as does CVS. We determined the nucleotide alterations of representative mutants from each group by using the dideoxy method of RNA sequencing. In the glycoproteins of eight nonpathogenic or attenuated mutants, we identified an amino acid substitution at position 333. Arginine 333 was replaced by either glutamine or glycine. In the glycoprotein of eight pathogenic mutants, we identified an amino acid substitution at lysine 330, asparagine 336, or isoleucine 338. Thus, although all substitutions affected neutralization and were located close to each other in the glycoprotein sequence, only substitutions at position 333 affected pathogenicity.