Effect of gene constellation and postreassortment amino acid change on the phenotypic features of H5 influenza virus reassortants

Reassortants between a low-pathogenic avian influenza virus strain A/Duck/Primorie/2621/2001 (H5N2) and a high-yield human influenza virus strain A/Puerto Rico/8/34 (H1N1) were generated, genotyped and analyzed with respect to their yield in embryonated chicken eggs, pathogenicity for mice, and immunogenicity. A reassortant having HA and NA genes from A/Duck/Primorie/2621/2001 virus and 6 genes from A/Puerto Rico/8/34 virus (6:2 reassortant) replicated efficiently in embryonated chicken eggs, the yields being intermediate between the yields of the avian parent virus and those of the A/Puerto Rico/8/34 parent strain. The reassortant having the HA gene from A/Duck/Primorie/2621/2001 virus and 7 genes from A/Puerto Rico/8/34 virus (7:1 reassortant) produced low yields. A variant of the 7:1 reassortant selected by serial passages in eggs had an amino acid substitution in the hemagglutinin (N244D, H3 numbering). The variant produced yields similar to those of the 6:2 reassortant. A 5:3 reassortant generated by a back-cross of the 6:2 reassortant with the avian parent and having PB1, HA and NA genes of A/Duck/Primorie/2621/2001 virus produced higher yields than the 7:1 or 6:2 reassortants, although still lower than the yields of A/Puerto Rico/8/34 virus. The 7:1, 6:2 and 5:3 reassortants were pathogenic for mice, with the level of virulence close to A/Puerto Rico/8/34 virus, in contrast to the extremely low pathogenicity of the A/Duck/Primorie/2621/2001 parent strain. Immunization of mice with an inactivated 6:2 H5N2 reassortant provided efficient immune protection against a reassortant virus containing the HA and NA genes of a recent H5N1 isolate. The results are discussed in connection with the problem of the improvement of vaccine strains against the threatening H5N1 pandemic.