The cold adapted and temperature sensitive influenza A/Ann Arbor/6/60 virus, the master donor virus for live attenuated influenza vaccines, has multiple defects in replication at the restrictive temperature

We have previously determined that the temperature sensitive (ts) and attenuated (att) phenotypes of the cold adapted influenza A/Ann Arbor/6/60 strain (MDV-A), the master donor virus for the live attenuated influenza A vaccines (FluMist (R)), are specified by the five amino acids in the PB1, PB2 and NP gene segments. To understand how these loci control the ts phenotype of MDV-A, replication of MDV-A at the non-permissive temperature (39 degrees C) was compared with recombinant wild-type A/Ann Arbor/6/60 (rWt). The mRNA and protein synthesis of MDV-A in the infected MDCK cells were not significantly reduced at 39 degrees C during a single-step replication, however, vRNA synthesis was reduced and the nuclear-cytoplasmic export of viral RNP (vRNP) was blocked. In addition, the virions released from MDV-A infected cells at 39 degrees C exhibited irregular morphology and had a greatly reduced amount of the M1 protein incorporated. The reduced M1 protein incorporation and vRNP export blockage correlated well with the virus ts phenotype because these defects could be partially alleviated by removing the three ts loci from the PB1 gene. The virions and vRNPs isolated from the MDV-A infected cells contained a higher level of heat shock protein 70 (Hsp70) than those of rWt, however, whether Hsp70 is involved in thermal inhibition of MDV-A replication remains to be determined. Our studies demonstrate that restrictive replication of MDV-A at the non-permissive temperature occurs in multiple steps of the virus replication cycle. (C) 2008 Elsevier Inc. All rights reserved.