EVALUATION OF THE GENETIC STABILITY OF THE TEMPERATURE-SENSITIVE PB2 GENE MUTATION OF THE INFLUENZA A ANN ARBOR/6/60 COLD-ADAPTED VACCINE VIRUS/

A single-gene reassortant bearing the PB2 gene of the A/Ann Arbor/6/60 cold-adapted virus in the background of the A/Korea/82 (H3N2) wild-ty pe virus is a temperature-sensitive (ts) virus with an in vitro shutof f temperature of 38 degrees C. A single mutation at amino acid (aa) at 265 (Asp-Ser) of the PB2 protein is responsible for the ts phenotype. This ts single-gene PB2 reassortant virus was serially passaged at el evated temperatures in Madin-Darby canine kidney cells to generate ts( +) phenotypic revertant viruses. Four ts(+) phenotypically revertant v iruses were derived independently, and each possessed a shutoff temper ature for replication in vitro of >40 degrees C. Each of the four phen otypically revertant viruses replicated efficiently in the upper and l ower respiratory tracts of mice and hamsters, unlike the PB2 single-ge ne reassortant virus, confirming that the ts phenotype was responsible for the attenuation of this virus in rodents. Mating the ts(+) revert ants with wild-type virus yielded ts progeny in high frequency, indica ting that the loss of ts phenotype was due to a suppressor mutation wh ich was mapped to the PA gene in each of the four independently derive d ts phenotypic revertants. Nucleotide sequence analysis confirmed the absence of new mutations on the PB2 gene and the presence of predicte d amino acid changes in the PA proteins of the revertant viruses. Thes e studies suggest that single amino acid changes at aa 245 (Glu-Lys) o r 337 (Asp-Asn) of the PA protein can completely suppress the ts and a ttenuation phenotypes specified by the Asp-Ser mutation at aa 265 of t he PB2 protein of the A/Ann Arbor/6/60 cold-adapted virus.