A COLD-PASSAGED, ATTENUATED STRAIN OF HUMAN RESPIRATORY SYNCYTIAL VIRUS CONTAINS MUTATIONS IN THE F-GENES AND L-GENES

In previous studies, a mutant (cp-RSV) of the RSV A2 strain derived fr om 52 serial cold passages in bovine embryonic tissue culture was high ly attenuated in seropositive adults and children but caused upper res piratory tract disease in seronegative infants. We investigated the ge neric basis for this attenuation phenotype by comparing the complete g enomic RNA sequence of this virus with the published sequence of strai n A2 as well as with that of its unattenuated wild-type parent (HEK-7) virus. RNA was extracted from virions grown in tissue culture, revers e transcribed, amplified by the polymerase chain reaction (PCR), clone d, and sequenced. Changes from the published A2 wild-type sequence wer e confirmed on independently derived cDNA clones and by direct sequenc ing of PCR fragments. The HEK-7 parent virus was then analyzed at thes e positions by direct sequencing of PCR fragments. Fifteen nucleotide differences between the published A2 wild-type virus and cp-RSV were f ound. None appeared to involve cis-acting RNA sequences. Of the 15 nuc leotide differences, only 1 occurred outside a translational open read ing frame (ORF), and 2 which did occur within ORFs were silent at the amino acid level. The remaining 12 nucleotide differences encoded amin o acid changes. All 3 of the mutations which were silent at the amino acid level, and 8 of the 12 which resulted in amino acid differences, were also present in the HEK-7 parent virus and therefore were not cha nges acquired during the cold passages. Thus, the remaining 4 nucleoti de differences and the attendant 4 amino acid changes are associated w ith the attenuation phenotype of the cp-RSV. Two of the changes occur in the F gene and two in the L gene. These results confirm the previou sly described RSV genomic sequence, provide the first sequence of a li ve attenuated RSV vaccine strain, provide the first sequence of an RSV strain which has been evaluated in chimpanzees and humans, and indica te that attenuation in humans of a pneumovirus can be associated with a relatively small number of nucleotide and amino acid changes.