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Seasonal inactivated influenza vaccines are usually trivalent or quadrivalent and are prepared from accredited seed viruses. Yields of influenza A seed viruses can be enhanced by gene reassortment with high-yielding donor strains, but similar approaches for influenza B seed viruses have been largely unsuccessful. For vaccine manufacture influenza B seed viruses are usually adapted for high-growth by serial passage. Influenza B antigen yields so obtained are often unpredictable and selection of influenza B seed viruses by this method can be a rate-limiting step in seasonal influenza vaccine manufacture. We recently have shown that selection of stable cold-adapted mutants from seasonal epidemic influenza B viruses is associated with improved growth. In this study, specific mutations were identified that were responsible for growth enhancement as a consequence of adaptation to growth at lower temperatures. Molecular analysis revealed that the following mutations in the HA, NP and NA genes are required for enhanced viral growth: G156/N160 in the HA, E253, G375 in the NP and T146 in the NA genes. These results demonstrate that the growth of seasonal influenza B viruses can be optimized or improved significantly by specific gene modifications.