Method for rapid generation of an attenuated RNA virus

A method for generating an attenuated RNA virus comprising:

• A) re-encoding the viral genome of an infectious RNA virus by randomly substituting a part of the nucleotide codons of the entire viral genome of said infectious RNA virus by another nucleotide codon encoding for the same amino acid,
• with the proviso that:
• i) the number and position of rare nucleotide codons present in said viral genome are not modified, said rare nucleotide codons being CGU, CGC, CGA, CGG, UCG, CCG, GCG and ACG;
• and
• ii) the regions of said viral genome which are involved with RNA secondary structure are not modified;
• B) generating an attenuated RNA virus by:
• i) introducing a promoter of DNA-dependent RNA polymerase in position 5′ and optionally a terminator and a RNA polyadenylation sequence in position 3′ of the re-encoded viral genome as obtained in step A;
• ii) amplifying the re-encoded viral genome as prepared in sub-step B) i) including said promoter and optionally said terminator and RNA polyadenylation sequence, in at least 2 overlapping cDNA fragments;
• iii) transfecting said cDNA fragments into a host cell;
• iv) incubating said host cell of sub-step B) iii);
• and
• v) recovering the attenuated RNA virus from said incubated host cell.