An influenza A live attenuated reassortant virus possessing three temperature-sensitive mutations in the PB2 polymerase gene rapidly loses temperature sensitivity following replication in hamsters

The purpose of the present study was to produce an influenza A H2N2 donor virus from which an attenuating PB2 gene bearing three discrete temperature sensitive (ts) mutations could be readily transferred to currently epidemic influenza A H1N1 and H3N2 viruses via genetic reassortment. An influenza A transfectant virus was first produced that contained site-directed ts mutations at amino acids 112, 265, and 556 in the PB2 gene of influenza A/AA/60 virus origin in a background of the other seven RNA segments fron the influenza A/LA/87 (H3N2) virus. The A/LA/87 PB2 is transfectant virus (clone 22B1) was mated with the A/AA/60 (H2N2) wild type virus, and six H2N2 is reassortants were obtained. One reassortant virus, clone 25Al, possessed the triple ts PB2 gene in the context of all seven other genes of homologous A/AA/60 origin. Isolation of this reassortment permitted an examination of the contribution of the ts PB2 transfectant virus to its attenuation and phenotypic stability independent from an effect of the A/AA/60-A/LA/87 gene constellation on attenuation, and less phenotypically stable than the A/LA/87 triple ts transfectant virus from which it was derived. The A/AA/60 reassortment possessing the PB2 gene containing three introduced ts mutations underwent rapid and significant loss of its temperature sensitivity following replication in the lungs of immunocompetent hamsters. This indicated that the A/AA/60-A/LA/87 gene constellation contributed significantly to the overall level of temperature-sensitivity, attenuation, and stability of the A/LA/87 triple is transfectant virus. It is likely that the instability of the ts phenotype exhibited by the A/AA/60 triple ts reassortment virus would not be acceptable for a vaccine to be used in humans. The implications of these findings for the usefulness of ts mutations as the sole attenuating mutation in influenza virus vaccines is discussed. Published by Elsevier Science Ltd.