The cold-adapted (ca) and temperature-sensitive (ts) influenza master donor virus (MDV) B/USSR/60/69 was derived from its wild-type parental virus after successive passages in eggs at 32 6C and 25 6C. This strain is currently in use for preparing reassortant influenza B vaccine viruses which are used in the Russian trivalent live attenuated influenza vaccine. Vaccine viruses are obtained by classical reassortment of MDV and a currently circulating wild-type virus. The phenotypic properties cold adaptation and temperature sensitivity are inherited from the six genes encoding the internal proteins of the MDV. However, the role of the individual gene segments in temperature sensitivity and thus attenuation is not known. In this study, 35 reassortant viruses of B/USSR/60/69 MDV with current wild-type non-ts influenza B viruses were generated in eggs or MDCK cells and studied in order to identify the genes responsible for their ts phenotype. For each virus the exact genome composition was determined as well as its ts phenotype. The results demonstrated that the polymerase PB2 and PA gene segments of B/USSR/60/69 MDV independently controlled expression of the ts phenotype of B/USSR/60/69 MDV-based reassortant viruses. The other genes coding for internal proteins played no role in this respect. This suggests that mutations in the polymerase genes PB2 and PA play an essential role in attenuation of B/USSR/60/69 MDV-based reassortant influenza B vaccine viruses.

Abstract-The cold adapted, temperature sensitive and attenuated influenza master donor viruses A/Lenin grad/134/17/57 (H2N2) and B/USSR/60/69 were used to generate vaccine viruses to be included in live attenuated influenza vaccine. These vaccine viruses typically are 6 : 2 reassortant viruses containing the gene segments of the surface antigens haemagglutinin and neuraminidase of current wild type influenza A and influenza B viruses with the gene segments encoding the internal viral proteins, conferring the cold adapted, temperature sensitive and attenuated phenotype, being inherited from the master donor viruses. The 6 : 2 reassortant viruses are selected from co infections between master donor virus and wild type viruses that the oretically may yield as many as 256 combinations of gene segments and thus 256 genetically different viruses. As the time to generate and isolate vaccine viruses is limited and because only 6 : 2 reassortant viruses are allowed as vaccine viruses, sboth selection and creening needs to be both rapid and unambiguous. The screen ing of reassortant viruses by RT PCRs using master donor virus and wild type virus specific primer sets is described to select both influenza A and influenza B 6 : 2 reassortant viruses to be used in seasonal and pan demic live attenuated vaccine, unambigously.