**4. Conclusion**

Understanding the gene constellation effect in influenza is important, especially for vaccine production. The mixing and matching of influenza genomic segments in nature and in the laboratory gives rise to new viruses with phenotypes that differ from the ancestral viruses. In nature, this may be a more pathogenic virus or one that has an expanded host range. In the laboratory, attenuated viruses with good growth characteristics and high protein yield are desirable for study and vaccine production. A greater understanding of what contributes to the gene constellation effect may enable researchers to produce influenza vaccine seed viruses that facilitate production with reduced risk of infection.

As we have described in this chapter, current research has provided some insight into the genomic features that contribute to the gene constellation effect, but more work needs to be done. Some segments, such as those encoding the glycoproteins and the polymerase proteins, appear to be more frequently involved in reassortments. The reassortment of the polymerase proteins is more common in laboratory manipulation whereas the reassort‐ ment of glycoproteins is more common in nature. The beneficial effects of certain pro‐ tein:protein interactions may be the underlying impetus behind some of these reassortments. For example, certain combinations of HA, NA and M can lead to changes in transmission and growth. Likewise, certain combinations of PB1, PB2 and PA can affect polymerase activity and growth. Also, the two smallest segments have effects on cell tropism and viral fitness. The amount of polymerase activity is not directly associated with virus titer suggesting other factors affecting replication must be balanced with replication efficiency. Changes in the polymerase segments can also affect pathogenicity, especially when the virus is adapting to a new host cell. Changes in the glycoproteins have also been shoen to affect pathogenicity. While much work has focused on either the glycoproteins or the replicase proteins independently, some of the work described here demonstrates that these two groups of proteins have an effect on each other. The interaction between these two groups of proteins at a functional level needs to be elucidated.

While several groups have analyzed the genomes of reassortant viruses there is still a great need for better understanding what features contribute to genomic reassortment. With more whole virus genome sequences available for analysis there is a better chance that the features important for reassortment can be determined. Retrospective analysis of reassor‐ tant viruses can illuminate which genomic features are compatible. In vitro construction of reassortant viruses can highlight which segments, or parts of segments, are not compati‐ ble. After a reassortment event is detected there needs to be more analyses of the muta‐ tions that occurred in each segment as they may have facilitated the reassortment event. Mutations necessary for reassortment would occur prior to reassortment and perhaps be present in bottleneck viruses. Mutations that occur with passage are those that increase the fitness of the reassortant. Description of both types of mutations would enhance our understanding of the network of interactions between viral proteins. In addition to the changes in coding sequences, analysis of the untranslated regions of the genomes is also important. There is no available information about the compatibility of segments with the replication and translation machinery, or how this contributes to the gene constellation.

Finally, an understanding of the gene constellation effect will allow for the selection of better reassortant viruses for vaccine production. Currently both the *in ovo* and reverse genetic methods use an impirical approach to get the best viruses that express the desired HA and NA proteins. Knowing how the different segments contribute to the network of interactions that result in high yield will enable researchers to produce strains that will provide the best backbone for an influenza vaccine seed virus. The optimal backbones may be universal, or differ for the different virus subtypes, or differ according to the host species that the virus providing the HA and NA infects. But without optimal virus backbones, the production of high yield reassortant influenza vaccine seed viruses will remain inefficient.
